WO2023109965A1 - Camptothecin compound and conjugate thereof - Google Patents

Camptothecin compound and conjugate thereof Download PDF

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WO2023109965A1
WO2023109965A1 PCT/CN2022/139765 CN2022139765W WO2023109965A1 WO 2023109965 A1 WO2023109965 A1 WO 2023109965A1 CN 2022139765 W CN2022139765 W CN 2022139765W WO 2023109965 A1 WO2023109965 A1 WO 2023109965A1
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structural formula
compound
compound shown
prodrug
pharmaceutically acceptable
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周伟
徐辉
朱会凯
王珍珍
谭小钉
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迈威(上海)生物科技股份有限公司
江苏迈威康新药研发有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/22—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the field of biotechnology, and more specifically, the invention relates to a new-type camptothecin analog and its application in the preparation of drugs, especially antibody-drug conjugates.
  • DNA topoisomerase is a class of essential enzymes widely present in organisms. It is a general term for enzymes that catalyze the mutual conversion of DNA topological isomers. It is mainly divided into topoisomerase I (Topoisomerase I, Topo I) and topoisomerase I. Constructase II (Topoisomerase II, Topo II) two types. Among them, topoisomerase I is highly expressed in a variety of tumor cells such as colon cancer, cervical cancer, and ovarian cancer, and its content is much higher than that of normal tissues or cells, and its activity is greatly increased in tumor cells in the S phase. Inhibitors of topoisomerase I activity can selectively inhibit DNA replication of proliferative tumor cells.
  • topoisomerase I is the main target of camptothecin (CPT) and its analogs.
  • Camptothecin is a cytotoxic quinoline alkaloid that stabilizes normally dissociated topoisomerase I and the covalent compound of the DNA strand to form a ternary complex. With the formation of the ternary complex, CPT inhibits the DNA cleavage and relinking reactions initially mediated by topoisomerase I, thereby inhibiting DNA synthesis, leading to cell death, and exerting anticancer effects.
  • Antibody-drug conjugate is a new type of tumor treatment drug composed of an antibody or antibody-like ligand, a small molecule drug, and a linker that couples the ligand to the drug. Combining the anti-tumor activity of small molecule drugs and the high selectivity, stability and good pharmacokinetic characteristics of antibodies or antibody-like ligands has become a hot spot in the field of tumor treatment.
  • the ADC drugs prepared by camptothecin (CPT) compounds at home and abroad mainly include:
  • ADC drugs have shown certain therapeutic effects, but there are also certain problems.
  • the half-life of IMMU-132 in plasma is only about 12h, which may lead to problems such as increased side effects, such as diarrhea, fatigue, nausea, febrile neutrophils, etc.
  • Different degrees of toxic reactions such as cytopenia and leukopenia (US2014/0170063A1).
  • the random coupling of DS-1062 and DS-7300 will result in greater heterogeneity of ADC products.
  • camptothecin compounds there are still relatively few types of ADCs containing camptothecin compounds, and there are disadvantages such as a relatively narrow range of target population, poor monotherapy effect, and strong side effects. Therefore, there is still a need to develop new camptothecin compounds and corresponding ADCs to meet the therapeutic needs.
  • the object of the present invention is to provide a compound with a new structure and an antibody-drug conjugate prepared therefrom.
  • the compound with the new structure is a camptothecin compound itself, or a camptothecin or a camptothecin compound A compound formed by linking a compound with a linker.
  • halogen means fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).
  • drug-containing linker refers to a compound obtained by directly or indirectly covalently bonding a drug (such as a small molecule drug, such as a camptothecin compound) to a linker.
  • a group when a group is substituted, it may be substituted by one or more substituents, the number of substituents being dependent on the number of hydrogen atoms contained in the group, all of which may be substituted.
  • the present invention provides camptothecin compounds.
  • the camptothecin compound is a compound represented by structural formula I or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
  • R 1 , R 2 , R 3 , R 4 are independently hydrogen, halogen, hydroxyl, C1-6 alkoxy, amino or substituted amino, C1 -7 alkyl or substituted C1-7 alkyl, or any two of R 1 , R 2 , R 3 , R 4 together with the carbon atoms they are connected to form a C3-6 cyclic alkyl group.
  • the C1-6 alkoxy groups include straight-chain or branched C1-6 alkoxy groups, preferably straight-chain Or a branched C1-3 alkoxy group, more preferably a methoxy group.
  • R 1 , R 2 , R 3 , and R 4 are independently substituted amino groups
  • the substituted amino groups are amino groups substituted by one or more substituents selected from methyl and ethyl.
  • R 1 , R 2 , R 3 , and R 4 are independently C1-7 alkyl or substituted C1-7 alkyl
  • the C1-7 alkyl or substituted C1-7 alkyl includes linear or branched Chain C1-7 alkyl or substituted C1-7 alkyl
  • the substituted C1-7 alkyl is C1-7 alkyl substituted by one or more substituents selected from cyclopropyl and cyclobutyl or
  • the straight or branched C1-7 alkyl or substituted C1-7 alkyl is preferably C1-3 alkyl or substituted C1-3 alkyl, such as methyl, halomethyl (preferably trifluoromethyl).
  • G is hydrogen, halogen, methyl or methoxy.
  • G is hydrogen, fluorine or chlorine.
  • Y is oxygen, sulfur, sulfone, sulfoxide, methylene or substituted methylene.
  • Substituted methylene can be that one hydrogen of methylene is substituted, also can be that two hydrogens are substituted simultaneously, and substituent can be benzyl or alkyl; When substituent is alkyl, described alkyl and R3 And/or R 4 and the carbon atoms connected to them can constitute a C3-6 membered ring or spiro ring structure.
  • the substituent of the substituted methylene group is preferably an alkyl group, more preferably a linear or branched C1-4 alkyl group.
  • Y is oxygen, sulfur, sulfone or sulfoxide; or, preferably, Y is oxygen, sulfur or methylene.
  • X is oxygen or sulfur
  • n 0 or 1.
  • R 1 , R 2 , R 3 , R 4 are independently hydrogen, halogen (such as fluorine), C1-7 alkyl or substituted C1-7 alkyl, or R 1 , R 2 , R 3 , R Any two of 4 together with the carbon atoms to which they are attached constitute a C3-6 cyclic alkyl group (for example a C3-5 cyclic alkyl group). Further, R 1 and R 2 may be the same; and/or, R 3 and R 4 may be the same.
  • Y is a methylene group substituted by an alkyl group, and the alkyl group, R 3 and/or R 4 and the carbon atoms connected to them may form a C3-6 membered ring or spiro ring structure.
  • X may be oxygen
  • X is oxygen
  • G is hydrogen
  • halogen eg fluorine or chlorine
  • Y and R 1 , R 2 , R 3 , R 4 are as defined above.
  • X is oxygen
  • G is hydrogen
  • Y is methylene or substituted methylene, oxygen or sulfur
  • R 1 , R 2 , R 3 , R 4 are as defined above.
  • X is oxygen
  • G is fluorine
  • Y is methylene or substituted methylene, oxygen or sulfur
  • R 1 , R 2 , R 3 , R 4 are as defined above.
  • X is oxygen
  • G is chlorine
  • Y is methylene or substituted methylene, oxygen or sulfur
  • R 1 , R 2 , R 3 , R 4 are as defined above.
  • X is oxygen
  • G is methyl
  • Y is methylene or substituted methylene, oxygen or sulfur
  • R 1 , R 2 , R 3 , R 4 are as defined above.
  • X is oxygen
  • G is methoxy
  • Y is methylene or substituted methylene, oxygen or sulfur
  • R 1 , R 2 , R 3 , R 4 are as defined above.
  • X is oxygen
  • G is hydrogen
  • Y is oxygen, sulfone or sulfoxide
  • R 1 , R 2 , R 3 , R 4 are as defined above.
  • X is oxygen
  • G is hydrogen
  • Y is sulfone or sulfoxide
  • R 1 , R 2 , R 3 , R 4 are as defined above.
  • the compound shown in the structural formula I provided by the present invention is further a compound shown in the structural formula IA:
  • the groups R 1 , R 2 , R 3 , R 4 have the same definitions as the groups R 1 , R 2 , R 3 , R 4 in the structural formula I above, but R 1 , R 2 , R 3 , R 4 is not simultaneously hydrogen.
  • the camptothecin compound is a compound represented by structural formula II or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
  • R is C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or substituted by C3-6 cyclic alkyl, phenyl or substituted phenyl substituted by one or more substituents.
  • R 5 is C1-5 alkyl or substituted C1-5 alkyl, said C1-5 alkyl includes straight or branched C1-5 alkyl. Further, R 5 is a C1-4 linear alkyl group.
  • R is a substituted C1-5 alkyl or a substituted C3-6 cyclic alkyl
  • the substituents are selected from the group consisting of halogen, hydroxyl, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3 -6 cyclic alkyl group; and wherein the substituted amino group is an amino group substituted by one or more substituents selected from methyl and ethyl.
  • R 5 is a substituted phenyl group
  • the substituent is selected from alkyl (such as C1-6 alkyl, preferably C1-3) or halogen.
  • G is hydrogen, halogen (eg fluorine), methyl or methoxy.
  • G is hydrogen, fluorine or chlorine.
  • X is oxygen or sulfur.
  • n 0 or 1.
  • the compound shown in the structural formula II provided by the present invention is further a compound shown in the structural formula IIA:
  • the group R 5 has the same definition as the group R 5 in formula II above, except that R 5 cannot be n-butyl.
  • the compound in the first aspect of the present invention, has the following structure:
  • the present invention provides a drug-containing linker having the structure shown in the general formula "L-A-CPT", wherein L represents a linker for an antibody-drug conjugate (ADC), A represents one or more amino acids, and CPT For camptothecin compounds.
  • L represents a linker for an antibody-drug conjugate (ADC)
  • A represents one or more amino acids
  • CPT For camptothecin compounds.
  • the drug-containing linker having a structure represented by general formula L-A-CPT is a compound represented by structural formula III or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
  • E is selected from the group wherein, Indicates the site of attachment to M:
  • E-1 E-2: E-3: E-4: E-5: and E-6:
  • M is phenylene or phenylene substituted by one or more substituents, or a chemical bond; in substituted phenylene, the substitution The group is selected from alkyl (such as C1-C6 alkyl, preferably C1-C4 alkyl), haloalkyl (such as halogenated C1-C6 alkyl, preferably halogenated C1-C4 alkyl, such as trifluoromethyl), alkyl Oxy (eg C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), halogen, ester, amido and cyano; preferably, M is halogen-substituted phenylene.
  • alkyl such as C1-C6 alkyl, preferably C1-C4 alkyl
  • haloalkyl such as halogenated C1-C6 alkyl, preferably halogenated C1-C4 alkyl, such as trifluoromethyl
  • SP 1 is selected from C1-8 alkylene, C1-8 cycloalkylene or C1-21 (preferably C1-16, more preferably C1-11 ) straight-chain heteroalkylene, the C1-21 straight-chain heteroalkylene contains 1-11 (preferably 1-6) heteroatoms selected from N, O or S, wherein the C1-8 alkylene
  • the group, C1-8 cycloalkylene and C1-21 linear heteroalkylene are each independently optionally substituted with one or more substituents selected from hydroxyl, amino, sulfonic acid and cyano.
  • SP is selected from -NH(CH2CH2O)aCH2CH2CO-, -NH(CH2CH2O)aCH2CO-, -S(CH2)aCO- or a chemical bond, wherein a is An integer of 1-20, preferably an integer of 1-10, more preferably an integer of 1-6.
  • A represents 2-4 amino acids.
  • A when A represents 2 amino acids, it can be NH-Phe-Lys-CO, NH-Val-Ala-CO, NH-Val-Lys-CO, NH-Ala-Lys-CO, NH-Val-Cit-CO , NH-Phe-Cit-CO, NH-Leu-Cit-CO, NH-Phe-Arg-CO or NH-Gly-Val-CO, preferably NH-Phe-Lys-CO, NH-Val-Ala-CO Or NH-Val-Cit-CO; when A represents 3 amino acids, it can be NH-Glu-Val-Ala-CO, NH-Glu-Val-Cit-CO or NH-Ala-Ala-Ala-CO, preferably NH-Glu-Val-Ala-CO or NH-Ala-Ala-Ala-CO; when A represents 4 amino acids, it can be NH-Phe-Lys-CO, NH-Val
  • CPT is a compound of the class of camptothecins.
  • R 6 , R 7 are independently hydrogen, halogen or Ar'S
  • Ar' is phenyl or phenyl substituted by one or more substituents, in In the substituted phenyl group, the substituent is selected from alkyl (such as C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (such as C1-C6 alkoxy, preferably C1-C4 alkoxy, Preference is given to methoxy), halogen, ester, amido and cyano.
  • Ar' is benzene
  • CPT is the compound shown in structural formula I or structural formula IA provided in the first aspect of the present invention above or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and corresponding specific compounds.
  • CPT is the compound shown in the structural formula I provided in the first aspect of the present invention above or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
  • the groups G, X, Y, R 1 , R 2 , R 3 , R 4 , n are the same as the groups G, X, Y, R 1 , R 2 , R in the structural formula I in the first aspect above 3 , R 4 , and n have the same definitions.
  • CPT is the compound shown in the structural formula IA provided in the first aspect of the present invention above or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
  • the groups R 1 , R 2 , R 3 , R 4 have the same definitions as the groups R 1 , R 2 , R 3 , R 4 in the structural formula IA in the first aspect above, but R 1 , R 2 , R 3 and R 4 can be hydrogen at the same time.
  • R6 and R7 can be hydrogen at the same time, or hydrogen at the same time.
  • R 1 , R 2 , R 3 , and R 4 in the structural formula IA may be hydrogen at the same time, or they may not be hydrogen at the same time.
  • R 1 , R 2 , R 3 , and R 4 in the structural formula IA are not hydrogen at the same time; when R 6 and R 7 are not hydrogen at the same time , R 1 , R 2 , R 3 , and R 4 in the structural formula IA may be hydrogen at the same time, or hydrogen at the same time.
  • the compound shown in Structural Formula I or Structural Formula IA is connected to the carboxyl group of A via its amino group (see Structural Formula I or Structural Formula IA respectively) through an amide bond, that is, the amino group of the compound shown in Structural Formula I or Structural Formula IA An amide bond is formed with the carboxyl group of A in formula III or IIIA.
  • CPT is the compound shown in structural formula II or structural formula IIA provided in the first aspect of the present invention above or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof , and the corresponding specific compounds.
  • CPT is the compound shown in the structural formula II provided in the first aspect of the present invention above or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
  • the groups G, R 5 , X, n are the same as the definitions of the groups G, R 5 , X, n in the structural formula II in the first aspect above.
  • CPT is the compound shown in the structural formula IIA provided in the first aspect of the present invention above or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
  • the group R 5 has the same definition as the group R 5 in formula IIA in the first aspect above, but may be n-butyl.
  • the compound shown in Structural Formula II or Structural Formula IIA is connected to the carboxyl group of A through its amino group (see Structural Formula II or Structural Formula IIA respectively) through an amide bond, that is, the amino group of the compound shown in Structural Formula II or Structural Formula IIA and The carboxyl group of A in structure III or IIIA forms an amide bond.
  • CPT in structural formula III and structural formula IIIA, can be an exteacan derivative shown in structural formula IV or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
  • R is hydrogen , trifluoromethyl, C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3- 6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, or halogen.
  • R is a substituted C1-5 alkyl or a substituted C3-6 cyclic alkyl
  • the substituents are selected from halogen, hydroxyl, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3-6 cyclic alkyl; and wherein the substituted amino group is an amino group substituted by one or more substituents selected from methyl and ethyl.
  • R6 and R7 can be hydrogen at the same time, or hydrogen at the same time.
  • R8 in Formula IV may or may not be hydrogen.
  • R6 and R7 when R6 and R7 are hydrogen at the same time, R8 may be hydrogen or not be hydrogen.
  • the compound shown in structural formula IV is connected to the carboxyl group of A via its hydroxyl group (see structural formula IV) that is connected to the same carbon as R 8 through a self-releasing structure, such as
  • the solid line indicates the linking position with the carboxyl group of A in the structural formula III or IIIA
  • the wavy line indicates the linking position with the hydroxyl group in the structural formula IV.
  • M is preferably phenylene or substituted phenylene
  • SP1 is C1-21 (preferably C1-16, more preferably C1- 11) Straight-chain heteroalkylene, which contains 1-11 (preferably 1-6) heteroatoms selected from N, O or S.
  • the compound shown in the structural formula III and the structural formula IIIA provided by the present invention is further a compound shown in the structural formula V.
  • R 6 and R 7 are independently Ar'S, and Ar' is a phenyl group or a phenyl group substituted by one or more substituents.
  • the substituents are selected from alkyl groups (such as C1-C6 Alkyl, preferably C1-C4 alkyl), alkoxy (eg C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), halogen, ester, amido and cyano.
  • Ar' is phenyl, 4-methylformyl substituted phenyl or 4-formylmorpholine substituted phenyl
  • Xh and Yh are independently hydrogen, halogen, haloalkyl (such as halogenated C1-C6 alkyl, preferably halogenated C1-C4 alkyl, such as trifluoro methyl) or alkoxy (eg C1-C6 alkoxy, preferably C1-C4 alkoxy, eg methoxy).
  • halogen such as halogenated C1-C6 alkyl, preferably halogenated C1-C4 alkyl, such as trifluoro methyl
  • alkoxy eg C1-C6 alkoxy, preferably C1-C4 alkoxy, eg methoxy
  • m is any integer from 1 to 10, preferably 1 to 5, more preferably 3-5.
  • A represents 2-4 amino acids, as defined above.
  • Structural Formula V the definition of CPT and its connection relationship in Structural Formula V are the same as CPT in Structural Formula III and Structural Formula IIIA, as defined above.
  • the compound shown in the structural formula V provided by the present invention is further a compound shown in the structural formula V-A:
  • the groups A, G, Y, R 1 , R 2 , R 3 , R 4 , X, n are the same as the groups A, G, Y, R 1 , R 2 , The definitions of R 3 , R 4 , X and n are the same.
  • G is hydrogen, fluorine or chlorine.
  • Y is methylene, sulfur or oxygen.
  • the compound shown in the structural formula V-A provided by the present invention is further a compound shown in the structural formula V-A-1:
  • the compound shown in the structural formula V provided by the present invention is further a compound shown in the structural formula V-B:
  • the groups A, G, R 5 , X, n are the same as the definitions of the groups A, G, R 5 , X, n in the above formula III or IIIA.
  • the compound represented by the structural formula V-B provided by the present invention is further a compound represented by the structural formula V-B-1:
  • the compound shown in the structural formula V provided by the present invention is further a compound shown in the structural formula V-C:
  • the groups A and R 8 have the same definitions as the groups A and R 8 in the above structural formula III or IIIV.
  • the compound shown in the structural formula V provided by the present invention is further a compound shown in the structural formula VI:
  • A represents 2-4 amino acids, as defined above.
  • CPT is a camptothecin compound.
  • the definition of CPT and its connection relationship in formula VI are the same as CPT in formula III and formula IIIA, as defined above.
  • the compound shown in the structural formula VI provided by the present invention is further a compound shown in the structural formula VI-A:
  • the groups A, G, Y, R 1 , R 2 , R 3 , R 4 , X, n are the same as the groups A, G, Y, R 1 , R in the above structural formula III or structural formula IIIA 2 , R 3 , R 4 , X, and n have the same definitions.
  • G is hydrogen, fluorine or chlorine.
  • Y is methylene, sulfur or oxygen.
  • the compound shown in the structural formula VI-A provided by the present invention is further a compound shown in the structural formula VI-A-1:
  • the groups A, G, R 5 , X, n are the same as the definitions of the groups A, G, R 5 , X, n in the above structural formula III or the structural formula IIIA.
  • the compound shown in structural formula VI-B is further a compound shown in structural formula VI-B-1:
  • the compound shown in the structural formula VI provided by the present invention is further a compound shown in the structural formula VI-C:
  • the definitions of the groups A and R 8 are the same as the definitions of the groups A and R 8 in the above structural formula III or the structural formula IIIA.
  • the structural formula VC can be further structured as shown in the structural formula VI-C.
  • the compound in the second aspect of the present invention, has the following structure:
  • the present invention provides a structure composed of the above structural formulas I, I-A, II, II-A, III, III-A, V, V-A, V-A-1, V-B, V-B-1, V-C, VI, VI-A, VI- Antibody drug conjugates prepared from compounds shown in A-1, VI-B, VI-B-1, VI-C or pharmaceutically acceptable salts, stereoisomers, solvates or prodrugs and antibodies or antibody fragments United things.
  • the antibody or antibody fragment targets a tumor-associated antigen such as HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1 (Mucin1), STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissuefactor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C (Guanylyl cyclase C), Mesothelin (Mesothelin), SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16
  • the antibody drug conjugate has the general formula In the structure shown, wherein mAb represents an antibody or an antibody fragment, the groups M, SP 1 , SP 2 , A and CPT are the same as those defined in the above structural formula III or structural formula IIIA for the groups M, SP 1 , SP 2 , A and CPT .
  • N is 1-10, preferably 1-8 (for example, 1-5), more preferably 3-8.
  • EL is selected from the following groups, wherein, Indicates that it is linked to cysteine in mAb, Indicates that it is connected to M:
  • E L -1a and/or EL -1b E L -2: E L -3: E L -4: E L -5: E L -6:
  • the mAb may be an IgG antibody or fragment thereof targeting any of the above tumor-associated antigens, preferably an IgG1 subtype antibody or fragment thereof.
  • the antibody-drug conjugate has a structure represented by the following general formula VII or general formula VIII:
  • N is 1-10, preferably 1-8 (eg 1-5), more preferably 3-8.
  • Ab corresponds to the above general formula mAbs in.
  • the definitions of the groups A and CPT are the same as the definitions of the groups A and CPT in the structural formula III or the structural formula IIIA provided in the second aspect above.
  • the antibody-drug conjugates represented by general formulas VII and VIII provided in the third aspect of the present invention can generate prodrug metabolites in cells (such as tumor cells).
  • the groups A and CPT have the same definitions as the groups A and CPT in the formula III or IIIA provided in the second aspect above.
  • the prodrug metabolite of the antibody drug conjugate prepared using the compound of formula VI-A is the compound shown in structural formula IX-A:
  • the groups A, G, Y, R 1 , R 2 , R 3 , R 4 , X, n are the same as the structural formula III provided in the second aspect above or the groups A, G, G, Y, R 1 , R 2 , R 3 , R 4 , X, and n have the same definitions.
  • the compound shown in the structural formula IX-A provided by the present invention is further a compound shown in the structural formula IX-A-1:
  • the prodrug metabolite of the antibody drug conjugate prepared using the compound of formula VI-B is a compound shown in structural formula IX-B:
  • the groups A, G, R 5 , X, n have the same definitions as the groups A, G, R 5 , X, n in the formula III or IIIA provided in the second aspect above.
  • the groups A and R8 have the same definitions as the groups A and R8 in the structural formula III or the structural formula IIIA provided in the second aspect above.
  • the present invention provides the use of the compound according to the present invention or its pharmaceutically acceptable salts, stereoisomers, solvates or prodrugs or antibody-drug conjugates in the preparation of drugs for treating tumors.
  • the tumor is cancer.
  • the tumor and tumor-associated antigens such as HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM , Mucin 1 (Mucin 1), STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor (Tissuefactor), c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C (Guanylyl cyclase C), Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Positive or high expression of Trop-2, CEACAM5,
  • the tumor is colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, liver cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, esophageal squamous Cell carcinoma, head and neck squamous cell carcinoma, melanoma, leukemia, lymphoma, glioma, glioblastoma.
  • the present invention provides a method for treating tumors using a compound according to the present invention or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug or an antibody drug conjugate, the method comprising administering the A subject in need thereof is administered the compound or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug or antibody drug conjugate thereof.
  • the tumor is cancer.
  • the tumor and tumor-associated antigens such as HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM , Mucin 1 (Mucin 1), STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor (Tissuefactor), c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C (Guanylyl cyclase C), Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Positive or high expression of Trop-2, CEACAM5,
  • the tumor is colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, liver cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, esophageal squamous Cell carcinoma, head and neck squamous cell carcinoma, melanoma, leukemia, lymphoma, glioma, glioblastoma.
  • the subject is a mammal, preferably a primate, more preferably a human.
  • FIG. 1 HIC analysis results of the antibody drug conjugate of the present invention.
  • Figure 2 The pharmacodynamic results of the antibody-drug conjugate of the present invention in a mouse model of pancreatic cancer.
  • Fig. 3 The pharmacodynamic results of the antibody-drug conjugate of the present invention in a mouse model of bladder cancer.
  • Fig. 4 The pharmacodynamic results of the antibody-drug conjugate of the present invention in a mouse model of lung cancer.
  • Fig. 7 Research results of the antibody-drug conjugates of the present invention inducing tumor cell apoptosis.
  • Fig. 8 The dose-effect curve of the binding of the antibody-drug conjugate and antibody of the present invention to tumor cells.
  • camptothecin compound of the present invention can be obtained by the Friedlander reaction of the compound shown in formula A and the CDE tricyclic compound, and the general reaction formula is as follows:
  • the CDE tricyclic compound was purchased from MCE:
  • HCDE tricyclic compounds were prepared according to Bioorganic and Medicinal Chemistry, 2010, vol.18, #9, p.3140-3146:
  • the aminolactam compound is prepared according to the method of the patent publication CN106349233A.
  • the diaminoethyl ester intermediate was dissolved in dichloromethane (200ml), triethylamine (20.2g, 0.2mol, 2eq) was added, the temperature was cooled to below 10°C in an ice bath, and acetic anhydride (25.5g, 0.25mol, 2.5 eq).
  • Compound A3 was synthesized according to the method of patent publication US2004266803A.
  • step 1
  • the A4-2 intermediate was prepared from the A4-1 compound according to the literature method.
  • step 1
  • step 1
  • the A1-4 compound (1.25g, 5mmol, 1eq) was dissolved in 150ml of tetrahydrofuran, cooled to -60°C, added LDA (2M in THF, 7.6ml, 15.2mmol, 3.04eq ), after the addition was completed, the reaction was stirred at -60°C for 30 minutes.
  • MeI (1.45g, 10.21mmol, 2.04eq) was added, after the addition was complete, the dry ice bath was removed, the temperature was raised naturally, and the reaction was carried out overnight at room temperature.
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • Embodiment group 1.2 camptothecin compounds synthesis (Friedlander reaction)
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • Embodiment 1.2.16 Synthesis of camptothecin compound 18
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • GGFG-Dxd is synthesized according to the method described in the patent publication (US20190151328A1):
  • the BL linker compound was synthesized according to the method described in the patent publication WO2018/095422A1:
  • BL linker compound (857mg, 1mmol), GGFG-Dxd (840mg, 1mmol, 1eq), DIPEA (323mg, 2.5mmol, 2.5eq), HATU (570mg, 1.5mmol, 1.5eq), dissolved in 30ml DCM, stirred reaction 2h.
  • the reaction solution was cooled to 5-10°C, 1N hydrochloric acid (20ml) was added, and stirred for 0.5h.
  • the layers were separated, the aqueous phase was extracted with DCM (30ml*2), and the organic phases were combined.
  • the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered with suction, and spin-dried.
  • BL linker compound (857mg, 1mmol), HoSu (138mg, 1.2mmol, 1.2eq), DCC (310mg, 1.5mmol, 1.5eq) were dissolved in 30ml DCM, stirred at room temperature for 3h, the reaction solution was suction filtered, and the filtrate was A- DCM solution of Osu. The filtrate was added to the mixed solution of the crude intermediate 2-2, DIPEA (323 mg, 2.5 mmol, 2.5 eq), and DCM (30 ml), and the reaction was stirred for 3 h. The reaction solution was cooled to 10°C, 1N hydrochloric acid (20ml) was added, and stirred for 0.5h.
  • Boc-Gly-OH (175mg, 1mmol), A1 compound (176mg, 1mmol, 1eq), DIPEA (322mg, 2.5mmol, 2.5eq), HATU (456mg, 1.2mmol, 1.2eq) were dissolved in 30ml DCM, and the reaction was stirred 2h.
  • BL linker compound (318mg, 0.37mmol), HoSu (51mg, 0.44mmol, 1.2eq), DCC (114mg, 0.56mmol, 1.5eq) were dissolved in 30ml DCM, stirred at room temperature for 3h, and the reaction solution was suction filtered. The filtrate was added to the mixed solution of the crude intermediate 3-4, DIPEA (120mg, 0.93mmol, 2.5eq), DCM (30ml), and stirred for 3h. The reaction solution was cooled to 10°C, 1N hydrochloric acid (20ml) was added, and stirred for 0.5h. The layers were separated, the aqueous phase was extracted with DCM (30ml*2), and the organic phases were combined.
  • the synthetic route is as follows:
  • BL linker compound (146mg, 0.17mmol), HoSu (25mg, 0.21mmol, 1.2eq), DCC (54mg, 0.26mmol, 1.5eq) were dissolved in 20ml DCM, stirred at room temperature for 3h, and the reaction solution was suction filtered. The filtrate was added to the mixed solution of the crude intermediate 5-1, DIPEA (56mg, 0.43mmol, 2.5eq), and DCM (20ml), and the reaction was stirred for 3h. The reaction solution was cooled to 5-10°C, 1N hydrochloric acid (20ml) was added, and stirred for 0.5h. The layers were separated, the aqueous phase was extracted with DCM (30ml*2), and the organic phases were combined.
  • Embodiment group 3 compares the synthesis of drug-containing linker
  • the synthetic route is as follows:
  • Linker L-C was synthesized according to the method similar to drug-containing linker L-1.
  • the antibody is reduced to open the disulfide bond, and then coupled with the linker to form a bridged antibody-drug conjugate.
  • Antibody reduction use Sephadex G25 carrier NAP-25 chromatographic column to replace 120mg antibody sample to pH 7.0 containing 50mM sodium chloride, 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, and the antibody The concentration was diluted to 10mg/ml. Take 10ml of antibody samples totaling 100mg, and add 10mg/ml TCEP (Sigma-Aldrich) aqueous solution at an antibody-TCEP molar ratio of 1:10 equivalent, and the volume of TCEP aqueous solution added is 2.1ml. After 2 hours of incubation, use the Sephadex G25 chromatographic column reaction solution to replace to a pH 6.5 buffer solution containing 50mM sodium chloride, 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer.
  • TCEP Sigma-Aldrich
  • the NAP-25 chromatographic column with Sephadex G25 carrier was used to replace the reaction solution to the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH 8.0 to remove the excess drug-containing linker, and heated in a water bath at 37°C for 3 hours.
  • Purification of antibody-drug conjugates Concentrate the above samples using AMICOM ultrafiltration centrifuge tubes, and concentrate the samples to about 15 mg/mL. Add 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate+3M ammonium phosphate buffer solution until the conductivity is 100ms/cm. Load the sample to TOYOPEAL Butyl-650M filler (purchased from TOSOH) hydrophobic column, phase A is 50mm disodium hydrogen phosphate-sodium dihydrogen phosphate + 0.6M ammonium sulfate, phase B is buffered with 50mm disodium hydrogen phosphate-sodium dihydrogen phosphate solution. Phase B was eluted with a 0-100% gradient of 8 column volumes, and the main peak was collected.
  • the concentration of the antibody-drug conjugate can be obtained by measuring the UV absorbance at 280 nm and the characteristic absorption wavelength of small molecules, and then performing the following calculations.
  • the concentration of the antibody-drug conjugate has the following relationship:
  • MW ADC is the molecular weight of the antibody-drug conjugate
  • MW Ab is the molecular weight of the antibody
  • MW D is the molecular weight of the drug-containing linker
  • Sample preparation Dilute the sample to 2.0mg/ml with mobile phase B, centrifuge at 12000rpm for 10min, and take the supernatant for HPLC analysis;
  • Mobile phase A 1.5M (NH4) 2 SO 4 +25mM PB, pH 7.0;
  • Mobile phase B 25mM PB+20%IPA, pH 7.0;
  • Sample preparation Dilute the sample to 2.0mg/ml with mobile phase B, centrifuge at 12000rpm for 10min, and take the supernatant for HPLC analysis;
  • Mobile phase B 25mM PB+20%IPA, pH 7.0;
  • Sample preparation Dilute the sample to 2.0mg/ml with mobile phase B, centrifuge at 12000rpm for 10min, and take the supernatant for HPLC analysis;
  • Sample treatment Take an appropriate amount of sample in an ultrafiltration tube, replace it with 50mM NH4HCO3 replacement buffer (pH7.1), add buffer, and ultrafiltration centrifuge (13000g*5min). Add 8 ⁇ l of PNGase F enzyme to the replaced sample, incubate at 37°C for 5 hours for desugaring treatment, after the incubation, centrifuge at 12000rpm for 5 minutes, take the supernatant into the injection vial as the test sample for use.
  • Mobile phase 50mM ammonium acetate, pH 7.0;
  • Atomizer pressure 20psig
  • Sheath gas temperature 325°C
  • Sheath gas flow 12L/min
  • Sample processing After diluting the sample to about 1.0 mg/ml with mobile phase, centrifuge at 12000 rpm for 10 min, and take the supernatant for sample analysis.
  • Sample pretreatment Process the sample according to the method described in the patent publication (US 10227417 B2).
  • each of the targeting Trop-2 antibody hTINA1 (prepared according to the sequence of Datopotamab in WHO Drug Information) and h23-12 were respectively combined with drug-containing linkers MWD-L1 and MWC- ADCs were prepared from L2, MWC-L3, MWF-L6, MWD-L7, MWD-L8, MWD-L9 and MWF-L8.
  • the bridged ADC drugs 1a, 1b, 1c, 1d, 1j, 1k, 1l, 1m, 1n, 1o, and 1p were obtained; the intermediate samples of 1d without hydrophobic chromatography were collected and saved as intermediate sample 1j.
  • each of the targeting Trop-2 antibodies hTINA1 and h23-12 were mixed with MC-GGFG-Dxd, L-A, L-B, L-J, MWS-L7, L-I and MWS-L6 respectively to prepare ADCs to obtain random coupling ADC 1e, 1f, 1g, 1h, 1i, 1q, 1r, 1s, 1t, and 1u.
  • the DAR value, concentration and Purity was measured.
  • reverse action chromatography 4.2f in group 4 of this embodiment to measure the DAR value of ADC samples (1e, 1h, 1q, 1r, 1s, 1t and 1u) containing MC-GGFG-Dxd drug-containing linker
  • reverse Chromatography 4.2g measures the DAR value of ADC samples (1f, 1g, 1i) containing L-A or L-B drug-containing linkers; use 4.2a ultraviolet spectrophotometry and 4.2d size exclusion chromatography in Example Group 4 to determine The concentration and purity of non-bridging ADC drugs (1e-1i) were determined.
  • h23-12 is a monoclonal antibody with the following sequence:
  • Heavy chain CDR1 SYWMH (SEQ ID NO: 1)
  • Heavy chain CDR2 EITPSDNYGSYNQKFKG (SEQ ID NO: 2)
  • Heavy chain CDR3 GHGNYVSFDY (SEQ ID NO: 3)
  • HER-2-targeting antibody trastuzumab (CAS: 180288-69-1, purchased from Shanghai Roche Pharmaceutical Co., Ltd.) was mixed with the drug-containing linker MWD -L1, MWD-L2, MWE-L4, MWF-L6, MWD-L8, MWD-L9, L-C, MWC-L3 were used to prepare bridging ADC 2a, 2b, 2c, 2h, 2i, 2d, 2l.
  • the DAR value, concentration and Purity was measured.
  • Her2-ADC-1 Trastuzumab MWC-L1 3.9 4.6 98.0% 2b
  • Her2-ADC-2 Trastuzumab MWC-L2 4.0 3.8 99.2%
  • Her2-ADC-3 Trastuzumab MWE-L4 4.0 3.7 97.5% 2d
  • Her2-ADC-4 Trastuzumab L-C 3.9 3.8 97.72
  • Her2-ADC-5 Trastuzumab L-A 4.3 4.8 99.0% 2f
  • Her2-ADC-6 Trastuzumab L-B 4.1 6.2 98.6% 2g
  • Her2-ADC-7 Trastuzumab MC-GGFG-Dxd 4.0 6.6 99.8% 2 hours
  • Her2-ADC-8 Trastuzumab MWD-L8 4.0 7.5 98.9% 2i
  • Her2-ADC-9 Trastuzumab MWD-L9 4.0 7.8
  • the Trop2 samples (1a, 1h, 1j) and Her2 samples (2b, 2e) prepared in Example 1 and Example 2 were analyzed by hydrophobic chromatography using 4.2b in Group 4 of this example as follows to compare the results of different connection techniques. Drug Heterogeneity of ADCs.
  • Her2-ADC-2 Comparing Her2-ADC-2 and Her2-ADC-5, it can be seen that the ADC drug Her2-ADC-2 prepared using the drug-containing linker MWC-L2 has better uniformity than Her2-ADC-5 prepared using the known linker L-A .
  • Oral epithelial cancer cells KB (purchased from: ATCC) were cultured in DMEM medium supplemented with 10% FBS; human gastric cancer cells NCI-N87 (purchased from: ATCC) were cultured in RPMI1640 medium supplemented with 10% FBS; human colon cancer cells HT29 (purchased from: From: ATCC) was cultured using RPMI1640 medium supplemented with 10% FBS; human pancreatic adenocarcinoma cell BxPC-3 (purchased from: ATCC) was cultured using RPMI1640 medium supplemented with 10% FBS.
  • the data is fitted with 4 parameters using prism7 software, and the maximum killing formula is: 1-(OD450 value of the maximum killing well-OD450 value of the culture base)/(OD450 value of the cell maximum growth well-OD450 value of the culture base)*100 %.
  • the results are shown in Table 4.
  • Her2-targeting ADC samples prepared above were diluted with the complete medium, sequentially diluted to 50ug/ml, and then diluted in a 4-fold gradient, with a total of 9 gradients plus a zero point, and three replicate wells were set for all samples.
  • Column 11 should set up a negative control (cells + medium) and a blank control (no cells, pure medium), and then place it in a cell incubator and incubate for 120 hours or 168 hours (see table for details). Take out the cell culture plate, add 40 ⁇ l/well of MTS, and react in a 37°C incubator for 2-4h; take out the cell plate, and read the OD value at 490nm.
  • the above-prepared ADC samples targeting Trop2 were diluted with the complete medium, sequentially diluted to 50ug/ml, and then 4-fold serially diluted, a total of 9 gradients plus a zero point, and three replicate wells were set for all samples.
  • Column 11 should set up a negative control (cells + medium) and a blank control (no cells, pure medium), and then place them in a cell incubator and incubate for 120 hours. Take out the cell culture plate, add 40 ⁇ l/well of MTS, and react in a 37°C incubator for 2-4h; take out the cell plate, and read the OD value at 490nm.
  • Example 5.4 In vitro pharmacodynamic study of different ADC toxins on low-abundance tumors and cells with low antigen expression near tumor cells
  • Cell HS-746T was purchased from ATCC; the cells were cultured with RPMI 1640/IMEM (1:1) culture medium containing 10% fetal bovine serum (FBS), and penicillin and streptomycin were added to the culture medium at the same time, at 37°C, containing 5 cultured in an incubator with % CO2 air.
  • RPMI 1640/IMEM (1:1) culture medium containing 10% fetal bovine serum (FBS), and penicillin and streptomycin were added to the culture medium at the same time, at 37°C, containing 5 cultured in an incubator with % CO2 air.
  • the cells were treated with 0.1 ⁇ g/mL and 1 ⁇ g/mL Trop2-ADC-1, Trop2-ADC-2, Trop2-ADC-5, Trop2-ADC-10, Trop2-ADC-14 respectively for 168 hours, Collect the cells and count them; then incubate the cells with the above-mentioned Trop2-ADC labeled with Dylight 488 NHS Ester at 4°C in the dark for 1 hour, centrifuge to remove the supernatant, resuspend in phosphate buffer (PBS, pH7.4), wash with PBS (pH7.4) was washed three times, and the number of Hs746t cells was calculated by flow cytometer BD ACCURI C6 PLUS.
  • PBS phosphate buffer
  • pH7.4 wash with PBS
  • KPL-4 cells and MDA-MB-468 cells were purchased from ATCC, and the cells were cultured with RPMI 1640/IMEM (1:1) culture medium containing 10% fetal bovine serum (FBS), and penicillin and streptomycin were added to the culture medium at the same time , cultured at 37°C in an incubator containing 5% CO2 air.
  • RPMI 1640/IMEM (1:1) culture medium containing 10% fetal bovine serum (FBS), and penicillin and streptomycin were added to the culture medium at the same time , cultured at 37°C in an incubator containing 5% CO2 air.
  • HER2-positive KPL-4 cells and HER2-negative MDA-MB-468 cells were co-inoculated, or HER2-negative MDA-MB-468 cells were inoculated alone, after 168 hours of drug treatment, the cells were collected, and the cells were mixed with Dylight 488 NHS Ester
  • the labeled anti-HER2 antibody was incubated at 4°C in the dark for 1 hour, centrifuged to remove the supernatant, resuspended in PBS (pH 7.4), washed three times with PBS, and calculated by flow cytometer BD ACCURI C6 PLUS to calculate KPL-4 and MDA- MB-468 cell ratio, and calculate the respective cell numbers.
  • each ADC group has better tumor inhibitory effect on negative cells.
  • the comparison of the number of positive cells and the number of negative cells in each concentration group can be known.
  • the bystander killing effect of MWF-L6 and MWC-L2 was better than that of the control group GGFG-DXD and L-B.
  • BxPc-3 cells Human pancreatic cancer BxPc-3 cells, which were purchased from the Cell Bank of the Chinese Academy of Sciences, were used. BxPc-3 cells were cultured in 10-cm culture dishes adherently. The culture conditions were RPMI 1640 medium plus 10% fetal bovine serum, penicillin and streptomycin, and cultured at 37°C in an incubator containing 5% CO2. Subculture 2-3 times a week, when the cells are in the exponential growth phase, trypsinize, collect the cells, count and inoculate.
  • the antibody-drug conjugates Trop2-ADC-1, Trop2-ADC-2, Trop2-ADC-3, and Trop2-ADC-5 containing different drug linkers were used to conduct group comparison studies under different dosage group conditions (Table 13).
  • the mice were administered intravenously (IV), with an administration volume of 10 mL/kg; the solvent group was administered the same volume of solvent (physiological saline); see Table 13 for the specific dosage and regimen.
  • the tumor volume was measured twice a week, the body weight of the mice was weighed, and the data were recorded.
  • HT1376 cells Human bladder cancer HT1376 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. HT1376 cells were cultured in 10-cm culture dishes adherently. The culture conditions were RPMI 1640 culture medium plus 10% fetal bovine serum, penicillin and streptomycin, and cultured at 37°C in an incubator containing 5% CO2. Subculture 2-3 times a week, when the cells are in the exponential growth phase, trypsinize, collect the cells, count and inoculate.
  • the lung cancer cell Calu-3 which was purchased from the Cell Bank of the Chinese Academy of Sciences, was used. Calu-3 cells were cultured in 10-cm culture dishes adherently. The culture conditions were RPMI 1640 culture medium plus 10% fetal bovine serum, penicillin and streptomycin, and cultured at 37°C in an incubator containing 5% CO2. Subculture 2-3 times a week, when the cells are in the exponential growth phase, trypsinize, collect the cells, count and inoculate.
  • the antibody-drug conjugate Trop2-ADC-14 was taken as an example, and a group comparison study was carried out on Trop2-ADC-14 under different dosage groups (Table 15).
  • the mice were administered intravenously (IV), with an administration volume of 10 mL/kg; the solvent group was administered the same volume of solvent (physiological saline); see Table 15 for the specific dosage and regimen.
  • the tumor volume was measured twice a week, the body weight of the mice was weighed, and the data were recorded.
  • Table 15 The curative effect of Trop2-ADC-14 on human lung cancer Calu-3 subcutaneously transplanted tumor in nude mice (TGI% calculated according to tumor volume).
  • TROP2-positive BxPC-3 cells purchased from ATCC
  • TROP2-negative HT-29 cells purchased from ATCC
  • TROP2-negative HT-29 cells were co-seeded or TROP2-negative HT-29 cells were seeded in a 6-well plate alone. After being treated with Trop2-ADC-14 (10, 30, 100 ng/mL) or DS-1062a (100, 300 ng/mL) for 144 hours, the cells were collected and counted.
  • Trop2-ADC-14 has a strong bystander effect, killing TROP2-positive cells and killing TROP2-negative cells at the same time; Trop2-ADC-14 has a bystander effect at 30 ng/mL that is comparable to that of the reference drug DS.
  • the effect of -1062a was comparable at 300ng/mL (5A in Figure 5), and the results suggested that the bystander effect of Trop2-ADC-14 was stronger than that of the reference drug DS-1062 as a whole.
  • Both Trop2-ADC-14 and DS-1062a had no significant effect on the proliferation of TROP2-negative HT-29 cells cultured alone (5B in Figure 5).
  • the SRB method was used to detect the effects of antibody-drug conjugates or camptothecin compounds on the proliferation of tumor cells cultured in vitro for adherent cells. Inoculate a certain number of cells in the logarithmic growth phase in a 96-well culture plate, and after growing overnight, add different concentrations of antibodies, antibody-drug conjugates or camptothecin compounds. After 144 hours, fix with trichloroacetic acid. After staining with SRB (prepared with 1% glacial acetic acid, concentration 4mg/mL), add 10mM Tris solution to each well to dissolve. Read the OD value with a microplate reader at a wavelength of 510 nm.
  • Inhibition rate (%) (OD value control well-OD value administration well)/OD value control well ⁇ 100%
  • MTT method was used to detect the effects of antibody-drug conjugates or camptothecin compounds on the proliferation of tumor cells cultured in vitro.
  • a certain number of cells in the logarithmic growth phase were inoculated in a 96-well culture plate, and after the adherent growth overnight, different concentrations of antibody-drug conjugates or camptothecin compounds were added.
  • MTT was added to each well, and culture was continued for 4 hours at 37° C. in a 5% CO2 saturated humidity incubator. Add 100 ⁇ L triple solution to each well, and measure the OD value at 570 nm and 690 nm wavelengths on a microplate reader.
  • BxPC-3 cells purchased from: ATCC
  • Trop2-ADC-14, Trop2-ADC-14mAb i.e. antibody h23-12
  • incubate 37°C 3, 6, 12, 18, 24 and 48 hours.
  • Trop2-ADC-14 and h23-12 were endocytosed by the cells after incubation with TROP2-positive cells BxPC-3; endocytosis was time-dependent, and the internalized drugs gradually increased with time; Trop2-ADC-14 internalized drug more than antibody h23-12 at 24 hours.
  • BxPC-3 cells (purchased from ATCC) were inoculated in 6-well plates, treated with Trop2-ADC-14 (0.020, 0.196, 1.963nM), DS-1062 (0.020, 0.196, 1.963nM), Trop2-ADC-14mAb (ie Antibody h23-12) (6.728nM) and compound 3 (0.1, 1, 10nM) were treated for 120 hours, the cells were collected, Annexin-V-FITC and PI were added, stained at room temperature for 15 minutes in the dark, and finally 300 ⁇ l 1x binding buffer was added Resuspend and detect apoptosis with a flow cytometer (BD AccuriTMC6 Plus flow cytometer), with 1 ⁇ 10 4 cells in each sample gate. The experimental data were analyzed with BD CSamplerTMPlus C6 Plus software. The results are shown in Figure 7.
  • Trop2-ADC-14 0.196nM can obviously induce the degradation of pro-PARP, a marker protein of apoptosis, and induce the hydrolysis of pro-caspase 3 into active protein caspase 3 (cleaved-caspase 3), and the apoptosis-inducing effect has concentration-dependent; the apoptosis-inducing effect of Trop2-ADC-14 cells was significantly stronger than that of DS-1062a at the same concentration, and the apoptosis-inducing effect of the latter was not obvious at 0.196nM; the small molecule toxin compound 3 also concentration-dependently induced BxPC- 3 cell apoptosis; antibody h23-12 had no obvious apoptosis effect on BxPC-3 cells.
  • Trop2-ADC-14 and antibody h23-12 bind to TROP2-positive tumor cell BxPC-3 in a concentration-dependent manner, and the binding EC50 is 1296.0 ⁇ 155.6ng/mL and 1137.5 ⁇ 128.0ng/mL, respectively, suggesting that both The binding ability is equivalent to that of TROP2-positive cells; as a control, Trop2-ADC-14, antibody h23-12, and TROP2-negative cells HT-29 have no obvious binding.

Abstract

Provided is a camptothecin compound, which is a compound represented by structural formula I or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof. An antibody-drug conjugate comprising the camptothecin compound is also provided.

Description

一种喜树碱类化合物及其偶联物A kind of camptothecin compound and its conjugate
相关申请的交叉引用Cross References to Related Applications
本专利申请要求于2021年12月16日提交的申请号为CN202111544686.7的中国发明专利申请的优先权权益，在此将其全部内容引入作为参考。This patent application claims the priority right of the Chinese invention patent application with application number CN202111544686.7 filed on December 16, 2021, the entire contents of which are hereby incorporated by reference.
技术领域technical field
本发明属于生物技术领域，更具体地，本发明涉及一种新型结构的喜树碱类似物及其在制备药物、特别是抗体药物偶联物中的应用。The invention belongs to the field of biotechnology, and more specifically, the invention relates to a new-type camptothecin analog and its application in the preparation of drugs, especially antibody-drug conjugates.
背景技术Background technique
DNA拓扑异构酶是广泛存在于生物体内的一类必需酶，是催化DNA拓扑学异构体相互转变的酶的总称，主要分为拓扑异构酶I(Topoisomerase I，Topo I)与拓扑异构酶II(Topoisomerase II，Topo II)这两类。其中，拓扑异构酶I在多种肿瘤细胞如结肠癌、宫颈癌、卵巢癌中有高表达，含量大大高于正常组织或细胞的含量，并且在S期肿瘤细胞中活性大幅提高，因此针对拓扑异构酶I的活性抑制剂可选择性抑制增殖期肿瘤细胞DNA复制。DNA topoisomerase is a class of essential enzymes widely present in organisms. It is a general term for enzymes that catalyze the mutual conversion of DNA topological isomers. It is mainly divided into topoisomerase I (Topoisomerase I, Topo I) and topoisomerase I. Constructase II (Topoisomerase II, Topo II) two types. Among them, topoisomerase I is highly expressed in a variety of tumor cells such as colon cancer, cervical cancer, and ovarian cancer, and its content is much higher than that of normal tissues or cells, and its activity is greatly increased in tumor cells in the S phase. Inhibitors of topoisomerase I activity can selectively inhibit DNA replication of proliferative tumor cells.
研究表明，拓扑异构酶I是喜树碱(Camptothecin，CPT)及其类似物的主要靶点。喜树碱是一种细胞毒性喹啉类生物碱，可使正常解离的拓扑异构酶I和DNA链的共价化合物保持稳定，形成三元复合物。随着三元复合物的形成，CPT抑制了最初由拓扑异构酶I介导的DNA裂解和重新链接反应，从而抑制DNA的合成，导致细胞死亡，发挥抗癌作用。Studies have shown that topoisomerase I is the main target of camptothecin (CPT) and its analogs. Camptothecin is a cytotoxic quinoline alkaloid that stabilizes normally dissociated topoisomerase I and the covalent compound of the DNA strand to form a ternary complex. With the formation of the ternary complex, CPT inhibits the DNA cleavage and relinking reactions initially mediated by topoisomerase I, thereby inhibiting DNA synthesis, leading to cell death, and exerting anticancer effects.
鉴于上述性质，喜树碱及其类似物已经成为一类重要的抗肿瘤药物；并且作为小分子药物用于抗体药物偶联物中。抗体药物偶联物(Antibody-drug conjugate，ADC)是由抗体或抗体类配体、小分子药物以及将该配体与药物偶联起来的连接子共同组成的一种新型肿瘤治疗药物，其结合了小分子药物的抗肿瘤活性和抗体或抗体类配体的高选择性、稳定性和良好的药代动力学特征，已经成为目前肿瘤治疗领域的关注热点。In view of the above properties, camptothecin and its analogs have become an important class of anti-tumor drugs; and are used in antibody-drug conjugates as small molecule drugs. Antibody-drug conjugate (Antibody-drug conjugate, ADC) is a new type of tumor treatment drug composed of an antibody or antibody-like ligand, a small molecule drug, and a linker that couples the ligand to the drug. Combining the anti-tumor activity of small molecule drugs and the high selectivity, stability and good pharmacokinetic characteristics of antibodies or antibody-like ligands has become a hot spot in the field of tumor treatment.
迄今，国内外使用喜树碱(CPT)类化合物制备的ADC药物主要有：So far, the ADC drugs prepared by camptothecin (CPT) compounds at home and abroad mainly include:
1.Sacituzumab govitecan(IMMU-132)，其是将拓扑异构酶I抑制剂SN38通过CL2A-SN38含药连接子与靶向Trop-2的SAC抗体偶联，制备得到DAR＝8的靶向Trop-2ADC药物。1. Sacituzumab govitecan (IMMU-132), which is prepared by coupling the topoisomerase I inhibitor SN38 with the SAC antibody targeting Trop-2 through the CL2A-SN38 drug-containing linker, and preparing the targeting Trop-2 with DAR=8 -2 ADC drugs.
Figure PCTCN2022139765-appb-000001
Figure PCTCN2022139765-appb-000001
2.第一三共的Enhertu(DS-0821)，其是将Exatecan通过MC-GGFG-Dxd含药连接子与靶向HER2抗体Trastuzumab偶联，制得DAR＝8的ADC药物。2. Daiichi Sankyo’s Enhertu (DS-0821), which couples Exatecan with the HER2-targeting antibody Trastuzumab through the MC-GGFG-Dxd drug-containing linker to obtain an ADC drug with DAR=8.
Figure PCTCN2022139765-appb-000002
Figure PCTCN2022139765-appb-000002
3.第一三共在研的DS-1062，其是将Exatecan通过MC-GGFG-Dxd含药连接子通过随机偶联方式与靶向Trop-2的hTINA1抗体偶联，制备得到DAR＝4的ADC药物；DS-7300，其是将MC-GGFG-Dxd含药连接子通过随机偶联方式与靶向B7H3的hM30抗体偶联，制备得到DAR＝4的靶向B7H3ADC药物。3. DS-1062 under research by Daiichi Sankyo, which is prepared by coupling Exatecan with the hTINA1 antibody targeting Trop-2 through MC-GGFG-Dxd drug-containing linker through random coupling to obtain DAR=4 ADC drug; DS-7300, which is a B7H3-targeted ADC drug with DAR=4 prepared by coupling MC-GGFG-Dxd drug-containing linker with hM30 antibody targeting B7H3 through random coupling.
Figure PCTCN2022139765-appb-000003
Figure PCTCN2022139765-appb-000003
以上ADC药物均显示出了一定的治疗效果，但也存在一定的问题。例如，由于IMMU-132中碳酸酯键的化学不稳定性，IMMU-132在血浆中半衰期仅为约12h，可能导致副作用增加等问题，患者会出现包括腹泻、疲劳、恶心、发热性中性粒细胞减少和白细胞减少等的不同程度毒性反应(US2014/0170063A1)。而DS-1062和DS-7300采用随机偶联的方式会造成ADC产物具有较大的异质性。The above ADC drugs have shown certain therapeutic effects, but there are also certain problems. For example, due to the chemical instability of the carbonate bond in IMMU-132, the half-life of IMMU-132 in plasma is only about 12h, which may lead to problems such as increased side effects, such as diarrhea, fatigue, nausea, febrile neutrophils, etc. Different degrees of toxic reactions such as cytopenia and leukopenia (US2014/0170063A1). However, the random coupling of DS-1062 and DS-7300 will result in greater heterogeneity of ADC products.
目前，含喜树碱类化合物的ADC的可选择种类仍比较少，而且存在目标人群范围比 较窄、单药治疗效果不佳、毒副作用强等不利之处。因此，仍需要开发新的喜树碱类化合物以及相应的ADC，以满足治疗需求。At present, there are still relatively few types of ADCs containing camptothecin compounds, and there are disadvantages such as a relatively narrow range of target population, poor monotherapy effect, and strong side effects. Therefore, there is still a need to develop new camptothecin compounds and corresponding ADCs to meet the therapeutic needs.
发明内容Contents of the invention
鉴于上述问题，本发明的目的是提供一种新结构的化合物以及采用其制备的抗体药物偶联物，该新结构的化合物为喜树碱类化合物本身，或者为喜树碱或喜树碱类化合物与连接子连接形成的化合物。In view of the above problems, the object of the present invention is to provide a compound with a new structure and an antibody-drug conjugate prepared therefrom. The compound with the new structure is a camptothecin compound itself, or a camptothecin or a camptothecin compound A compound formed by linking a compound with a linker.
在本发明的上下文中，卤素是指氟(F)、氯(Cl)、溴(Br)或碘(I)。In the context of the present invention, halogen means fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).
在本发明的上下文中，“接头”与“连接子”可互换使用。In the context of the present invention, "linker" and "linker" are used interchangeably.
在本发明的上下文中，术语“含药连接子”是指药物(例如小分子药物，诸如喜树碱类化合物)与连接子直接或间接以共价键键合得到的化合物。In the context of the present invention, the term "drug-containing linker" refers to a compound obtained by directly or indirectly covalently bonding a drug (such as a small molecule drug, such as a camptothecin compound) to a linker.
在本发明的上下文中，当基团被取代时，其可以由一个或多个取代基取代，取代基的数目取决于基团所含的氢原子数目，全部氢原子均可被取代。In the context of the present invention, when a group is substituted, it may be substituted by one or more substituents, the number of substituents being dependent on the number of hydrogen atoms contained in the group, all of which may be substituted.
本发明的技术方案如下。The technical scheme of the present invention is as follows.
第一方面，本发明提供喜树碱类化合物。In a first aspect, the present invention provides camptothecin compounds.
在本发明的第一方面，所述喜树碱类化合物为结构式I所示的化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药：In the first aspect of the present invention, the camptothecin compound is a compound represented by structural formula I or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
Figure PCTCN2022139765-appb-000004
Figure PCTCN2022139765-appb-000004
在结构式I中，并且在本发明的上下文中如无其他说明，R 1、R 2、R 3、R 4独立地为氢、卤素、羟基、C1-6烷氧基、氨基或取代氨基、C1-7烷基或取代的C1-7烷基，或者R 1、R 2、R 3、R 4中的任意两个连同它们所连接的碳原子构成C3-6环状烷基。当R 1、R 2、R 3、R 4独立地为C1-6烷氧基时，所述C1-6烷氧基包括直链或支链的C1-6烷氧基，优选地为直链或支链的C1-3烷氧基，更优选甲氧基。当R 1、R 2、R 3、R 4独立地为取代氨基时，所述取代氨基为由选自甲基和乙基的一个或多个取代基取代的氨基。当R 1、R 2、R 3、R 4独立地为C1-7烷基或取代的C1-7烷基时，所述C1-7烷基或取代的C1-7烷基包括直 链或支链的C1-7烷基或取代的C1-7烷基，并且，取代的C1-7烷基为由选自环丙基和环丁基的一个或多个取代基取代的C1-7烷基；或者，所述直链或支链的C1-7烷基或取代的C1-7烷基优选为C1-3烷基或取代的C1-3烷基，例如甲基、卤代甲基(优选三氟甲基)。 In formula I, and unless otherwise stated in the context of the present invention, R 1 , R 2 , R 3 , R 4 are independently hydrogen, halogen, hydroxyl, C1-6 alkoxy, amino or substituted amino, C1 -7 alkyl or substituted C1-7 alkyl, or any two of R 1 , R 2 , R 3 , R 4 together with the carbon atoms they are connected to form a C3-6 cyclic alkyl group. When R 1 , R 2 , R 3 , and R 4 are independently C1-6 alkoxy groups, the C1-6 alkoxy groups include straight-chain or branched C1-6 alkoxy groups, preferably straight-chain Or a branched C1-3 alkoxy group, more preferably a methoxy group. When R 1 , R 2 , R 3 , and R 4 are independently substituted amino groups, the substituted amino groups are amino groups substituted by one or more substituents selected from methyl and ethyl. When R 1 , R 2 , R 3 , and R 4 are independently C1-7 alkyl or substituted C1-7 alkyl, the C1-7 alkyl or substituted C1-7 alkyl includes linear or branched Chain C1-7 alkyl or substituted C1-7 alkyl, and the substituted C1-7 alkyl is C1-7 alkyl substituted by one or more substituents selected from cyclopropyl and cyclobutyl or, the straight or branched C1-7 alkyl or substituted C1-7 alkyl is preferably C1-3 alkyl or substituted C1-3 alkyl, such as methyl, halomethyl (preferably trifluoromethyl).
在结构式I中，并且在本发明的上下文中如无其他说明，G为氢、卤素、甲基或甲氧基。优选地，G为氢、氟或氯。In formula I, and unless stated otherwise in the context of the present invention, G is hydrogen, halogen, methyl or methoxy. Preferably, G is hydrogen, fluorine or chlorine.
在结构式I中，并且在本发明的上下文中如无其他说明，Y为氧、硫、砜、亚砜、亚甲基或取代亚甲基。取代亚甲基可以是亚甲基的一个氢被取代，也可以是两个氢同时被取代，取代基可以为苄基或烷基；当取代基为烷基时，所述烷基与R 3和/或R 4以及连同它们所连接的碳原子可以构成C3-6元并环或螺环的结构。当Y为取代亚甲基时，取代亚甲基的取代基优选为烷基，更优选为直链或支链的C1-4烷基。优选地，Y为氧、硫、砜或亚砜；或者，优选地，Y为氧、硫或亚甲基。 In formula I, and unless stated otherwise in the context of the present invention, Y is oxygen, sulfur, sulfone, sulfoxide, methylene or substituted methylene. Substituted methylene can be that one hydrogen of methylene is substituted, also can be that two hydrogens are substituted simultaneously, and substituent can be benzyl or alkyl; When substituent is alkyl, described alkyl and R3 And/or R 4 and the carbon atoms connected to them can constitute a C3-6 membered ring or spiro ring structure. When Y is a substituted methylene group, the substituent of the substituted methylene group is preferably an alkyl group, more preferably a linear or branched C1-4 alkyl group. Preferably, Y is oxygen, sulfur, sulfone or sulfoxide; or, preferably, Y is oxygen, sulfur or methylene.
在结构式I中，并且在本发明的上下文中如无其他说明，X为氧或硫。In formula I, and unless stated otherwise in the context of the present invention, X is oxygen or sulfur.
在结构式I中，并且在本发明的上下文中如无其他说明，n＝0或1。In formula I, and unless stated otherwise in the context of the present invention, n=0 or 1.
结构式I中，在R 1、R 2、R 3、R 4同时为氢、X为氧、n＝0的情况下，当Y为亚甲基时，G不可为氢或氟；而当Y为氧或硫时，G不可为氢。 In structural formula I, when R 1 , R 2 , R 3 , and R 4 are simultaneously hydrogen, X is oxygen, and n=0, when Y is methylene, G cannot be hydrogen or fluorine; and when Y is In the case of oxygen or sulfur, G cannot be hydrogen.
优选地，R 1、R 2、R 3、R 4独立地为氢、卤素(例如氟)、C1-7烷基或取代的C1-7烷基，或者R 1、R 2、R 3、R 4中的任意两个连同它们所连接的碳原子构成C3-6环状烷基(例如C3-5环状烷基)。进一步地，R 1、R 2可以是相同的；和/或，R 3、R 4可以是相同的。 Preferably, R 1 , R 2 , R 3 , R 4 are independently hydrogen, halogen (such as fluorine), C1-7 alkyl or substituted C1-7 alkyl, or R 1 , R 2 , R 3 , R Any two of 4 together with the carbon atoms to which they are attached constitute a C3-6 cyclic alkyl group (for example a C3-5 cyclic alkyl group). Further, R 1 and R 2 may be the same; and/or, R 3 and R 4 may be the same.
优选地，Y为由烷基取代的亚甲基，所述烷基与R 3和/或R 4以及连同它们所连接的碳原子可以构成C3-6元并环或螺环的结构。 Preferably, Y is a methylene group substituted by an alkyl group, and the alkyl group, R 3 and/or R 4 and the carbon atoms connected to them may form a C3-6 membered ring or spiro ring structure.
优选地，X可以为氧。Preferably, X may be oxygen.
优选地，X为氧，G为氢、卤素(例如氟或氯)、甲基或甲氧基，Y和R 1、R 2、R 3、R 4如上文所定义。 Preferably, X is oxygen, G is hydrogen, halogen (eg fluorine or chlorine), methyl or methoxy, Y and R 1 , R 2 , R 3 , R 4 are as defined above.
优选地，X为氧，G为氢，Y为亚甲基或取代亚甲基、氧或硫，R 1、R 2、R 3、R 4如上文所定义。 Preferably, X is oxygen, G is hydrogen, Y is methylene or substituted methylene, oxygen or sulfur, and R 1 , R 2 , R 3 , R 4 are as defined above.
优选地，X为氧，G为氟，Y为亚甲基或取代亚甲基、氧或硫，R 1、R 2、R 3、R 4如上文所定义。 Preferably, X is oxygen, G is fluorine, Y is methylene or substituted methylene, oxygen or sulfur, and R 1 , R 2 , R 3 , R 4 are as defined above.
优选地，X为氧，G为氯，Y为亚甲基或取代亚甲基、氧或硫，R 1、R 2、R 3、R 4如上文所定义。 Preferably, X is oxygen, G is chlorine, Y is methylene or substituted methylene, oxygen or sulfur, and R 1 , R 2 , R 3 , R 4 are as defined above.
优选地，X为氧，G为甲基，Y为亚甲基或取代亚甲基、氧或硫，R 1、R 2、R 3、R 4如上文所定义。 Preferably, X is oxygen, G is methyl, Y is methylene or substituted methylene, oxygen or sulfur, and R 1 , R 2 , R 3 , R 4 are as defined above.
优选地，X为氧，G为甲氧基，Y为亚甲基或取代亚甲基、氧或硫，R 1、R 2、R 3、 R 4如上文所定义。 Preferably, X is oxygen, G is methoxy, Y is methylene or substituted methylene, oxygen or sulfur, and R 1 , R 2 , R 3 , R 4 are as defined above.
优选地，X为氧，G为氢，Y为氧、砜或亚砜，R 1、R 2、R 3、R 4如上文所定义。 Preferably, X is oxygen, G is hydrogen, Y is oxygen, sulfone or sulfoxide, and R 1 , R 2 , R 3 , R 4 are as defined above.
优选地，X为氧，G为氢，Y为砜或亚砜，R 1、R 2、R 3、R 4如上文所定义。 Preferably, X is oxygen, G is hydrogen, Y is sulfone or sulfoxide, and R 1 , R 2 , R 3 , R 4 are as defined above.
优选地，本发明提供的结构式I所示的化合物进一步为结构式IA所示的化合物：Preferably, the compound shown in the structural formula I provided by the present invention is further a compound shown in the structural formula IA:
Figure PCTCN2022139765-appb-000005
Figure PCTCN2022139765-appb-000005
结构式IA中，基团R 1、R 2、R 3、R 4与上文结构式I中基团R 1、R 2、R 3、R 4的定义相同，但是R 1、R 2、R 3、R 4不同时为氢。 In the structural formula IA, the groups R 1 , R 2 , R 3 , R 4 have the same definitions as the groups R 1 , R 2 , R 3 , R 4 in the structural formula I above, but R 1 , R 2 , R 3 , R 4 is not simultaneously hydrogen.
或者，在本发明的第一方面，所述喜树碱类化合物为结构式II所示的化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药：Alternatively, in the first aspect of the present invention, the camptothecin compound is a compound represented by structural formula II or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
Figure PCTCN2022139765-appb-000006
Figure PCTCN2022139765-appb-000006
在结构式II中，并且在本发明的上下文中如无其他说明，R 5为C1-5烷基或由一个或多个取代基取代的C1-5烷基、C3-6环状烷基或由一个或多个取代基取代的C3-6环状烷基、苯基或取代苯基。当R 5为C1-5烷基或取代的C1-5烷基时，所述C1-5烷基包括直链或支链的C1-5烷基。进一步地，R 5为C1-4直链烷基。当R 5为取代的C1-5烷基或取代的C3-6环状烷基时，取代基选自卤素、羟基、甲氧基、三氟甲基、氨基或取代氨基、甲磺酰基和C3-6环状烷基；并且其中，取代氨基为由选自甲基和乙基的一个或多个取代基取代的氨基。当R 5为取代苯基时，取代基选自烷基(例如C1-6烷基、优选C1-3)或卤素。 In formula II, and unless otherwise stated in the context of the present invention, R is C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or substituted by C3-6 cyclic alkyl, phenyl or substituted phenyl substituted by one or more substituents. When R 5 is C1-5 alkyl or substituted C1-5 alkyl, said C1-5 alkyl includes straight or branched C1-5 alkyl. Further, R 5 is a C1-4 linear alkyl group. When R is a substituted C1-5 alkyl or a substituted C3-6 cyclic alkyl, the substituents are selected from the group consisting of halogen, hydroxyl, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3 -6 cyclic alkyl group; and wherein the substituted amino group is an amino group substituted by one or more substituents selected from methyl and ethyl. When R 5 is a substituted phenyl group, the substituent is selected from alkyl (such as C1-6 alkyl, preferably C1-3) or halogen.
在结构式II中，并且在本发明的上下文中如无其他说明，G为氢、卤素(例如氟)、甲基或甲氧基。优选地，G为氢、氟或氯。In formula II, and unless stated otherwise in the context of the present invention, G is hydrogen, halogen (eg fluorine), methyl or methoxy. Preferably, G is hydrogen, fluorine or chlorine.
结构式II中，X为氧或硫。In structural formula II, X is oxygen or sulfur.
结构式II中，n＝0或1。In the structural formula II, n=0 or 1.
结构式II中，当X为氧、G为氢、n＝0时，R 5不可为正丁基。 In the structural formula II, when X is oxygen, G is hydrogen, and n=0, R 5 cannot be n-butyl.
优选地，本发明提供的结构式II所示的化合物进一步为结构式IIA所示的化合物：Preferably, the compound shown in the structural formula II provided by the present invention is further a compound shown in the structural formula IIA:
Figure PCTCN2022139765-appb-000007
Figure PCTCN2022139765-appb-000007
结构式IIA中，基团R 5与上文结构式II中基团R 5的定义相同，但是R 5不可为正丁基。 In formula IIA, the group R 5 has the same definition as the group R 5 in formula II above, except that R 5 cannot be n-butyl.
根据本发明的具体实施方式，在本发明的第一方面，所述化合物具有如下结构：According to a specific embodiment of the present invention, in the first aspect of the present invention, the compound has the following structure:
Figure PCTCN2022139765-appb-000008
Figure PCTCN2022139765-appb-000008
Figure PCTCN2022139765-appb-000009
Figure PCTCN2022139765-appb-000009
Figure PCTCN2022139765-appb-000010
Figure PCTCN2022139765-appb-000010
第二方面，本发明提供具有通式“L-A-CPT”所示结构的含药连接子，其中L表示用于抗体药物偶联物(ADC)的连接子，A表示一个或多个氨基酸，CPT为喜树碱类化合物。In the second aspect, the present invention provides a drug-containing linker having the structure shown in the general formula "L-A-CPT", wherein L represents a linker for an antibody-drug conjugate (ADC), A represents one or more amino acids, and CPT For camptothecin compounds.
在本发明的第二方面，所述具有通式L-A-CPT所示结构的含药连接子为结构式III所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药。In the second aspect of the present invention, the drug-containing linker having a structure represented by general formula L-A-CPT is a compound represented by structural formula III or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
Figure PCTCN2022139765-appb-000011
Figure PCTCN2022139765-appb-000011
在结构式III中，并且在本发明的上下文中如无其他说明，E选自如下基团，其中，
Figure PCTCN2022139765-appb-000012
表示与M的连接位点： In formula III, and unless otherwise stated in the context of the present invention, E is selected from the group wherein,
Figure PCTCN2022139765-appb-000012
Indicates the site of attachment to M:
E-1：
Figure PCTCN2022139765-appb-000013
E-2：
Figure PCTCN2022139765-appb-000014
E-3：
Figure PCTCN2022139765-appb-000015
E-4：
Figure PCTCN2022139765-appb-000016
E-5：
Figure PCTCN2022139765-appb-000017
和E-6：
Figure PCTCN2022139765-appb-000018
E-1:
Figure PCTCN2022139765-appb-000013
E-2:
Figure PCTCN2022139765-appb-000014
E-3:
Figure PCTCN2022139765-appb-000015
E-4:
Figure PCTCN2022139765-appb-000016
E-5:
Figure PCTCN2022139765-appb-000017
and E-6:
Figure PCTCN2022139765-appb-000018
在结构式III中，并且在本发明的上下文中如无其他说明，M为亚苯基或由一个或多个取代基取代的亚苯基，或化学键；在取代的亚苯基中，所述取代基选自烷基(例如C1-C6烷基、优选C1-C4烷基)、卤代烷基(例如卤代C1-C6烷基、优选卤代C1-C4烷基，例如三氟甲基)、烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，优选甲氧基)、卤素、 酯基、酰胺基和氰基；优选地，M为卤素取代的亚苯基。In formula III, and unless otherwise stated in the context of the present invention, M is phenylene or phenylene substituted by one or more substituents, or a chemical bond; in substituted phenylene, the substitution The group is selected from alkyl (such as C1-C6 alkyl, preferably C1-C4 alkyl), haloalkyl (such as halogenated C1-C6 alkyl, preferably halogenated C1-C4 alkyl, such as trifluoromethyl), alkyl Oxy (eg C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), halogen, ester, amido and cyano; preferably, M is halogen-substituted phenylene.
在结构式III中，并且在本发明的上下文中如无其他说明，SP 1选自C1-8亚烷基、C1-8亚环烷基或C1-21(优选C1-16、更优选C1-11)直链亚杂烷基，所述C1-21直链亚杂烷基包含1-11个(优选1-6个)选自N、O或S的杂原子，其中所述C1-8亚烷基、C1-8亚环烷基和C1-21直链亚杂烷基各自独立地任选被选自羟基、氨基、磺酸基和氰基的一个或多个取代基取代。 In formula III, and unless otherwise stated in the context of the present invention, SP 1 is selected from C1-8 alkylene, C1-8 cycloalkylene or C1-21 (preferably C1-16, more preferably C1-11 ) straight-chain heteroalkylene, the C1-21 straight-chain heteroalkylene contains 1-11 (preferably 1-6) heteroatoms selected from N, O or S, wherein the C1-8 alkylene The group, C1-8 cycloalkylene and C1-21 linear heteroalkylene are each independently optionally substituted with one or more substituents selected from hydroxyl, amino, sulfonic acid and cyano.
在结构式III中，并且在本发明的上下文中如无其他说明，SP 2选自-NH(CH2CH2O)aCH2CH2CO-、-NH(CH2CH2O)aCH2CO-、-S(CH2)aCO-或化学键，其中a为1-20的整数，优选1-10的整数，更优选1-6的整数。 In formula III, and unless otherwise stated in the context of the present invention, SP is selected from -NH(CH2CH2O)aCH2CH2CO-, -NH(CH2CH2O)aCH2CO-, -S(CH2)aCO- or a chemical bond, wherein a is An integer of 1-20, preferably an integer of 1-10, more preferably an integer of 1-6.
在结构式III中，并且在本发明的上下文中如无其他说明，A表示2-4个氨基酸。其中，A表示2个氨基酸时，可以为NH-Phe-Lys-CO、NH-Val-Ala-CO、NH-Val-Lys-CO、NH-Ala-Lys-CO、NH-Val-Cit-CO、NH-Phe-Cit-CO、NH-Leu-Cit-CO、NH-Phe-Arg-CO或NH-Gly-Val-CO，优选为NH-Phe-Lys-CO、NH-Val-Ala-CO或NH-Val-Cit-CO；A表示3个氨基酸时，可以为NH-Glu-Val-Ala-CO、NH-Glu-Val-Cit-CO或NH-Ala-Ala-Ala-CO，优选为NH-Glu-Val-Ala-CO或NH-Ala-Ala-Ala-CO；A表示4个氨基酸时，可以为NH-Gly-Gly-Phe-Gly-CO或NH-Gly-Phe-Gly-Gly-CO，优选为NH-Gly-Gly-Phe-Gly-CO。优选地，A为NH-Val-Ala-CO、NH-Gly-Gly-Phe-Gly-CO或NH-Ala-Ala-Ala-CO。In formula III, and unless otherwise stated in the context of the present invention, A represents 2-4 amino acids. Among them, when A represents 2 amino acids, it can be NH-Phe-Lys-CO, NH-Val-Ala-CO, NH-Val-Lys-CO, NH-Ala-Lys-CO, NH-Val-Cit-CO , NH-Phe-Cit-CO, NH-Leu-Cit-CO, NH-Phe-Arg-CO or NH-Gly-Val-CO, preferably NH-Phe-Lys-CO, NH-Val-Ala-CO Or NH-Val-Cit-CO; when A represents 3 amino acids, it can be NH-Glu-Val-Ala-CO, NH-Glu-Val-Cit-CO or NH-Ala-Ala-Ala-CO, preferably NH-Glu-Val-Ala-CO or NH-Ala-Ala-Ala-CO; when A represents 4 amino acids, it can be NH-Gly-Gly-Phe-Gly-CO or NH-Gly-Phe-Gly-Gly -CO, preferably NH-Gly-Gly-Phe-Gly-CO. Preferably, A is NH-Val-Ala-CO, NH-Gly-Gly-Phe-Gly-CO or NH-Ala-Ala-Ala-CO.
在结构式III中，并且在本发明的上下文中如无其他说明，CPT为喜树碱类化合物。In formula III, and unless otherwise stated in the context of the present invention, CPT is a compound of the class of camptothecins.
其中，当基团E为E1时，本发明提供的结构式III进一步为结构式IIIA：Wherein, when the group E is E1, the structural formula III provided by the present invention is further a structural formula IIIA:
Figure PCTCN2022139765-appb-000019
Figure PCTCN2022139765-appb-000019
在结构式IIIA中，并且在本发明的上下文中如无其他说明，R 6、R 7独立地为氢、卤素或Ar’S，Ar’为苯基或由一个或多个取代基取代的苯基，在取代的苯基中，所述取代基选自烷基(例如C1-C6烷基、优选C1-C4烷基)、烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，优选甲氧基)、卤素、酯基、酰胺基和氰基。优选地，Ar’为苯 In formula IIIA, and unless otherwise stated in the context of the present invention, R 6 , R 7 are independently hydrogen, halogen or Ar'S, Ar' is phenyl or phenyl substituted by one or more substituents, in In the substituted phenyl group, the substituent is selected from alkyl (such as C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (such as C1-C6 alkoxy, preferably C1-C4 alkoxy, Preference is given to methoxy), halogen, ester, amido and cyano. Preferably, Ar' is benzene
基、4-甲基甲酰基取代苯基
Figure PCTCN2022139765-appb-000020
或4-甲酰基吗啉取代苯基
Figure PCTCN2022139765-appb-000021
base, 4-methylformyl substituted phenyl
Figure PCTCN2022139765-appb-000020
or 4-formylmorpholine substituted phenyl
Figure PCTCN2022139765-appb-000021
例如，在结构式III和结构式IIIA中，CPT为本发明上文第一方面中提供的结构式I或结构式IA所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，以及对应的具体化合物。For example, in structural formula III and structural formula IIIA, CPT is the compound shown in structural formula I or structural formula IA provided in the first aspect of the present invention above or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and corresponding specific compounds.
当CPT为本发明上文第一方面中提供的结构式I所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药时：When CPT is the compound shown in the structural formula I provided in the first aspect of the present invention above or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
Figure PCTCN2022139765-appb-000022
Figure PCTCN2022139765-appb-000022
结构式I中，基团G、X、Y、R 1、R 2、R 3、R 4、n与上文第一方面中结构式I中基团G、X、Y、R 1、R 2、R 3、R 4、n的定义相同。 In the structural formula I, the groups G, X, Y, R 1 , R 2 , R 3 , R 4 , n are the same as the groups G, X, Y, R 1 , R 2 , R in the structural formula I in the first aspect above 3 , R 4 , and n have the same definitions.
当CPT为本发明上文第一方面中提供的结构式IA所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药时：When CPT is the compound shown in the structural formula IA provided in the first aspect of the present invention above or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
Figure PCTCN2022139765-appb-000023
Figure PCTCN2022139765-appb-000023
结构式IA中，基团R 1、R 2、R 3、R 4与上文第一方面中结构式IA中基团R 1、R 2、R 3、R 4的定义相同，但是R 1、R 2、R 3、R 4可以同时为氢。 In the structural formula IA, the groups R 1 , R 2 , R 3 , R 4 have the same definitions as the groups R 1 , R 2 , R 3 , R 4 in the structural formula IA in the first aspect above, but R 1 , R 2 , R 3 and R 4 can be hydrogen at the same time.
特别地，当结构式IIIA所示化合物中的“CPT”为结构式IA所示化合物或其药学可接受的盐、立体异构体、溶剂化物或前药时，R 6和R 7可以同时为氢，或不同时为氢。同样地，结构式IA中的R 1、R 2、R 3、R 4可以同时为氢，或不同时为氢。根据本发明的具体实施方式，当R 6和R 7同时为氢时，结构式IA中的R 1、R 2、R 3、R 4不同时为氢；当 R 6和R 7不同时为氢时，结构式IA中的R 1、R 2、R 3、R 4可以同时为氢，或不同时为氢。 In particular, when "CPT" in the compound represented by structural formula IIIA is a compound represented by structural formula IA or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, R6 and R7 can be hydrogen at the same time, or hydrogen at the same time. Likewise, R 1 , R 2 , R 3 , and R 4 in the structural formula IA may be hydrogen at the same time, or they may not be hydrogen at the same time. According to a specific embodiment of the present invention, when R 6 and R 7 are hydrogen at the same time, R 1 , R 2 , R 3 , and R 4 in the structural formula IA are not hydrogen at the same time; when R 6 and R 7 are not hydrogen at the same time , R 1 , R 2 , R 3 , and R 4 in the structural formula IA may be hydrogen at the same time, or hydrogen at the same time.
在结构式III和结构式IIIA中，结构式I或结构式IA所示的化合物经由其氨基(分别参见结构式I或结构式IA)与A的羧基通过酰胺键相连，即在结构式I或结构式IA所示化合物的氨基与结构式III或IIIA中A的羧基形成了酰胺键。In Structural Formula III and Structural Formula IIIA, the compound shown in Structural Formula I or Structural Formula IA is connected to the carboxyl group of A via its amino group (see Structural Formula I or Structural Formula IA respectively) through an amide bond, that is, the amino group of the compound shown in Structural Formula I or Structural Formula IA An amide bond is formed with the carboxyl group of A in formula III or IIIA.
又如，在结构式III和结构式IIIA中，CPT为本发明上文第一方面中提供的结构式II或结构式IIA所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，以及对应的具体化合物。As another example, in structural formula III and structural formula IIIA, CPT is the compound shown in structural formula II or structural formula IIA provided in the first aspect of the present invention above or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof , and the corresponding specific compounds.
当CPT为本发明上文第一方面中提供的结构式II所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药时：When CPT is the compound shown in the structural formula II provided in the first aspect of the present invention above or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
Figure PCTCN2022139765-appb-000024
Figure PCTCN2022139765-appb-000024
结构式II中，基团G、R 5、X、n与上文第一方面中结构式II中基团G、R 5、X、n的定义相同。 In the structural formula II, the groups G, R 5 , X, n are the same as the definitions of the groups G, R 5 , X, n in the structural formula II in the first aspect above.
当CPT为本发明上文第一方面中提供的结构式IIA所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药时：When CPT is the compound shown in the structural formula IIA provided in the first aspect of the present invention above or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
Figure PCTCN2022139765-appb-000025
Figure PCTCN2022139765-appb-000025
结构式IIA中，基团R 5与上文第一方面中结构式IIA中基团R 5的定义相同，但是可以为正丁基。 In formula IIA, the group R 5 has the same definition as the group R 5 in formula IIA in the first aspect above, but may be n-butyl.
在结构式III和结构式IIIA中，结构式II或结构式IIA所示的化合物经由其氨基(分别参见结构式II或结构式IIA)与A的羧基通过酰胺键连接，即结构式II或结构式IIA所示化合物的氨基与结构式III或IIIA中A的羧基形成酰胺键。In Structural Formula III and Structural Formula IIIA, the compound shown in Structural Formula II or Structural Formula IIA is connected to the carboxyl group of A through its amino group (see Structural Formula II or Structural Formula IIA respectively) through an amide bond, that is, the amino group of the compound shown in Structural Formula II or Structural Formula IIA and The carboxyl group of A in structure III or IIIA forms an amide bond.
上文提出的化合物1、2、3、4、5、6、7、8、9、10、11、12、13、14、14-P、15、16、17、18、19、20、21、22、23、24、25、26、27、31、32、33、34、35、36、37、 38、39、40或其药学可接受的盐、立体异构体、溶剂化物或前药均可用作结构式III中的“CPT”，并通过其氨基与结构式III或IIIA中A的羧基通过酰胺键连接。 Compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14-P, 15, 16, 17, 18, 19, 20, 21 presented above , 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof Both can be used as "CPT" in the structural formula III, and the carboxyl group of A in the structural formula III or IIIA is connected through its amino group through an amide bond.
又如，在结构式III和结构式IIIA中，CPT可以为结构式IV所示的依喜替康(exteacan)衍生物或其药学可接受的盐、立体异构体、溶剂化物或前药：As another example, in structural formula III and structural formula IIIA, CPT can be an exteacan derivative shown in structural formula IV or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
Figure PCTCN2022139765-appb-000026
Figure PCTCN2022139765-appb-000026
在结构式IV中，并且在本发明的上下文中如无其他说明，R 8为氢、三氟甲基、C1-5烷基或由一个或多个取代基取代的C1-5烷基、C3-6环状烷基或由一个或多个取代基取代的C3-6环状烷基、或卤素。 In formula IV, and unless otherwise stated in the context of the present invention, R is hydrogen , trifluoromethyl, C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3- 6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, or halogen.
当R 8为取代的C1-5烷基或取代的C3-6环状烷基时，所述取代基选自卤素、羟基、甲氧基、三氟甲基、氨基或取代氨基、甲磺酰基和C3-6环状烷基；并且其中，取代氨基为由选自甲基和乙基的一个或多个取代基取代的氨基。 When R is a substituted C1-5 alkyl or a substituted C3-6 cyclic alkyl, the substituents are selected from halogen, hydroxyl, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3-6 cyclic alkyl; and wherein the substituted amino group is an amino group substituted by one or more substituents selected from methyl and ethyl.
特别地，当结构式IIIA所示化合物中的“CPT”为结构式IV所示化合物或其药学可接受的盐、立体异构体、溶剂化物或前药时，R 6和R 7可以同时为氢，或不同时为氢。同样地，结构式IV中的R 8可以为氢，或不为氢。根据本发明的具体实施方式，在R 6和R 7同时为氢时，R 8可以为氢或不为氢。 In particular, when "CPT" in the compound of structural formula IIIA is a compound of structural formula IV or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, R6 and R7 can be hydrogen at the same time, or hydrogen at the same time. Likewise, R8 in Formula IV may or may not be hydrogen. According to a specific embodiment of the present invention, when R6 and R7 are hydrogen at the same time, R8 may be hydrogen or not be hydrogen.
在结构式III和结构式IIIA中，结构式IV所示的化合物经由其与R 8连接于同一个碳的羟基(参见结构式IV)与A的羧基通过自释放结构相连，该自释放结构例如
Figure PCTCN2022139765-appb-000027
Figure PCTCN2022139765-appb-000028
实线表示与结构式III或结构式IIIA中A的羧基连接的位点，波浪线表示与结构式IV中羟基连接的位点。 In structural formula III and structural formula IIIA, the compound shown in structural formula IV is connected to the carboxyl group of A via its hydroxyl group (see structural formula IV) that is connected to the same carbon as R 8 through a self-releasing structure, such as
Figure PCTCN2022139765-appb-000027
Figure PCTCN2022139765-appb-000028
The solid line indicates the linking position with the carboxyl group of A in the structural formula III or IIIA, and the wavy line indicates the linking position with the hydroxyl group in the structural formula IV.
特别地，对于结构式IIIA所示的化合物，当R 6、R 7为Ar’S时，M优选为亚苯基或取代的亚苯基，同时SP1为C1-21(优选C1-16、更优选C1-11)直链亚杂烷基，所述直链亚杂烷基包含1-11个(优选1-6个)选自N、O或S的杂原子。 Especially, for the compound shown in structural formula IIIA, when R 6 and R 7 are Ar'S, M is preferably phenylene or substituted phenylene, and SP1 is C1-21 (preferably C1-16, more preferably C1- 11) Straight-chain heteroalkylene, which contains 1-11 (preferably 1-6) heteroatoms selected from N, O or S.
优选地，本发明提供的结构式III和结构式IIIA所示的化合物进一步为结构式V所 示的化合物。Preferably, the compound shown in the structural formula III and the structural formula IIIA provided by the present invention is further a compound shown in the structural formula V.
Figure PCTCN2022139765-appb-000029
Figure PCTCN2022139765-appb-000029
结构式V中，R 6、R 7独立地为Ar’S，Ar’为苯基或由一个或多个取代基取代的苯基，在取代的苯基中，取代基选自烷基(例如C1-C6烷基、优选C1-C4烷基)、烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，优选甲氧基)、卤素、酯基、酰胺基和氰基。优选地，Ar’为苯基、4-甲基甲酰基取代苯基
Figure PCTCN2022139765-appb-000030
或4-甲酰基吗啉取代苯基
Figure PCTCN2022139765-appb-000031
In the structural formula V, R 6 and R 7 are independently Ar'S, and Ar' is a phenyl group or a phenyl group substituted by one or more substituents. In the substituted phenyl group, the substituents are selected from alkyl groups (such as C1-C6 Alkyl, preferably C1-C4 alkyl), alkoxy (eg C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), halogen, ester, amido and cyano. Preferably, Ar' is phenyl, 4-methylformyl substituted phenyl
Figure PCTCN2022139765-appb-000030
or 4-formylmorpholine substituted phenyl
Figure PCTCN2022139765-appb-000031
结构式V中，并且在本发明的上下文中如无其他说明，Xh和Yh独立地为氢、卤素、卤代烷基(例如卤代C1-C6烷基、优选卤代C1-C4烷基，例如三氟甲基)或烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，例如甲氧基)。In formula V, and unless otherwise stated in the context of the present invention, Xh and Yh are independently hydrogen, halogen, haloalkyl (such as halogenated C1-C6 alkyl, preferably halogenated C1-C4 alkyl, such as trifluoro methyl) or alkoxy (eg C1-C6 alkoxy, preferably C1-C4 alkoxy, eg methoxy).
结构式V中，并且在本发明的上下文中如无其他说明，m为1～10、优选1～5、更优选3-5中任一整数。In formula V, and unless otherwise specified in the context of the present invention, m is any integer from 1 to 10, preferably 1 to 5, more preferably 3-5.
结构式V中，A表示2-4个氨基酸，如上文所定义。In formula V, A represents 2-4 amino acids, as defined above.
结构式V中，CPT的定义及其在结构式V中的连接关系与结构式III和结构式IIIA中CPT相同，如上文所定义。In Structural Formula V, the definition of CPT and its connection relationship in Structural Formula V are the same as CPT in Structural Formula III and Structural Formula IIIA, as defined above.
优选地，本发明提供的结构式V所示的化合物进一步为结构式V-A所示的化合物：Preferably, the compound shown in the structural formula V provided by the present invention is further a compound shown in the structural formula V-A:
Figure PCTCN2022139765-appb-000032
Figure PCTCN2022139765-appb-000032
Figure PCTCN2022139765-appb-000033
Figure PCTCN2022139765-appb-000033
结构式V-A中，基团A、G、Y、R 1、R 2、R 3、R 4、X、n与上文结构式III或结构式IIIA中基团A、G、Y、R 1、R 2、R 3、R 4、X、n的定义相同。 In the structural formula VA, the groups A, G, Y, R 1 , R 2 , R 3 , R 4 , X, n are the same as the groups A, G, Y, R 1 , R 2 , The definitions of R 3 , R 4 , X and n are the same.
优选地，结构式V-A中，G为氢、氟或氯。Preferably, in the structural formula V-A, G is hydrogen, fluorine or chlorine.
优选地，结构式V-A中，Y为亚甲基、硫或氧。Preferably, in the structural formula V-A, Y is methylene, sulfur or oxygen.
结构式V-A中，并且在本发明的上下文中如无其他说明，“-A-NH-”表示所示氨基与A中的羧基形成酰胺键。In formula V-A, and unless otherwise stated in the context of the present invention, "-A-NH-" means that the indicated amino group forms an amide bond with the carboxyl group in A.
优选地，本发明提供的结构式V-A所示的化合物进一步为结构式V-A-1所示的化合物：Preferably, the compound shown in the structural formula V-A provided by the present invention is further a compound shown in the structural formula V-A-1:
Figure PCTCN2022139765-appb-000034
Figure PCTCN2022139765-appb-000034
或者，本发明提供的结构式V所示的化合物进一步为结构式V-B所示的化合物：Alternatively, the compound shown in the structural formula V provided by the present invention is further a compound shown in the structural formula V-B:
Figure PCTCN2022139765-appb-000035
Figure PCTCN2022139765-appb-000035
结构式V-B中，基团A、G、R 5、X、n与上文结构式III或结构式IIIA中基团A、G、R 5、X、n的定义相同。 In the formula VB, the groups A, G, R 5 , X, n are the same as the definitions of the groups A, G, R 5 , X, n in the above formula III or IIIA.
结构式V-B中，并且在本发明的上下文中如无其他说明，“-A-NH-”表示所示氨基与A中的羧基形成酰胺键。In formula V-B, and unless otherwise stated in the context of the present invention, "-A-NH-" means that the indicated amino group forms an amide bond with the carboxyl group in A.
优选地，本发明提供的结构式V-B所示的化合物进一步为结构式V-B-1所示的化合物：Preferably, the compound represented by the structural formula V-B provided by the present invention is further a compound represented by the structural formula V-B-1:
Figure PCTCN2022139765-appb-000036
Figure PCTCN2022139765-appb-000036
或者，本发明提供的结构式V所示的化合物进一步为结构式V-C所示的化合物：Alternatively, the compound shown in the structural formula V provided by the present invention is further a compound shown in the structural formula V-C:
Figure PCTCN2022139765-appb-000037
Figure PCTCN2022139765-appb-000037
结构式V-C中，基团A、R 8与上文结构式III或结构式IIIV中基团A、R 8的定义相同。 In the structural formula VC, the groups A and R 8 have the same definitions as the groups A and R 8 in the above structural formula III or IIIV.
结构式V-C中，并且在本发明的上下文中如无其他说明，“-A-NH-”表示所示氨基与A中的羧基形成酰胺键。In formula V-C, and unless otherwise stated in the context of the present invention, "-A-NH-" means that the indicated amino group forms an amide bond with the carboxyl group in A.
结构式V-C中，并且在本发明的上下文中如无其他说明，
Figure PCTCN2022139765-appb-000038
与上文结构式III或结构式IIIV中的CPT为结构式IV时的自释放结构相同。 In the formula VC, and unless otherwise stated in the context of the present invention,
Figure PCTCN2022139765-appb-000038
It is the same as the self-release structure when the CPT in the above structural formula III or structural formula IIIV is the structural formula IV.
根据本发明的具体实施方式，本发明提供的结构式V所示的化合物进一步为结构式VI所示的化合物：According to the specific embodiment of the present invention, the compound shown in the structural formula V provided by the present invention is further a compound shown in the structural formula VI:
Figure PCTCN2022139765-appb-000039
Figure PCTCN2022139765-appb-000039
结构式VI中，A表示2-4个氨基酸，如上文所定义。In formula VI, A represents 2-4 amino acids, as defined above.
结构式VI中，CPT为喜树碱类化合物。CPT的定义及其在结构式VI中的连接关系与结构式III和结构式IIIA中CPT相同，如上文所定义。In the structural formula VI, CPT is a camptothecin compound. The definition of CPT and its connection relationship in formula VI are the same as CPT in formula III and formula IIIA, as defined above.
优选地，本发明提供的结构式VI所示的化合物进一步为结构式VI-A所示的化合物：Preferably, the compound shown in the structural formula VI provided by the present invention is further a compound shown in the structural formula VI-A:
Figure PCTCN2022139765-appb-000040
Figure PCTCN2022139765-appb-000040
结构式VI-A中，基团A、G、Y、R 1、R 2、R 3、R 4、X、n与上文结构式III或结构式IIIA中基团A、G、Y、R 1、R 2、R 3、R 4、X、n的定义相同。 In the structural formula VI-A, the groups A, G, Y, R 1 , R 2 , R 3 , R 4 , X, n are the same as the groups A, G, Y, R 1 , R in the above structural formula III or structural formula IIIA 2 , R 3 , R 4 , X, and n have the same definitions.
优选地，结构式VI-A中，G为氢、氟或氯。Preferably, in structural formula VI-A, G is hydrogen, fluorine or chlorine.
优选地，结构式VI-A中，Y为亚甲基、硫或氧。Preferably, in structural formula VI-A, Y is methylene, sulfur or oxygen.
结构式VI-A中，并且在本发明的上下文中如无其他说明，“-A-NH-”表示所示氨基与A中的羧基形成酰胺键。In Formula VI-A, and unless otherwise stated in the context of the present invention, "-A-NH-" means that the indicated amino group forms an amide bond with the carboxyl group in A.
优选地，本发明提供的结构式VI-A所示的化合物进一步为结构式VI-A-1所示的化合物：Preferably, the compound shown in the structural formula VI-A provided by the present invention is further a compound shown in the structural formula VI-A-1:
Figure PCTCN2022139765-appb-000041
Figure PCTCN2022139765-appb-000041
或者，结构式VI所示的化合物进一步为结构式VI-B所示的化合物：Or, the compound shown in structural formula VI is further the compound shown in structural formula VI-B:
Figure PCTCN2022139765-appb-000042
Figure PCTCN2022139765-appb-000042
结构式VI-B中，基团A、G、R 5、X、n与上文结构式III或结构式IIIA中基团A、G、R 5、X、n的定义相同。 In the structural formula VI-B, the groups A, G, R 5 , X, n are the same as the definitions of the groups A, G, R 5 , X, n in the above structural formula III or the structural formula IIIA.
结构式VI-B中，并且在本发明的上下文中如无其他说明，“-A-NH-”表示所示CPT中的氨基与A中的羧基形成酰胺键。In Formula VI-B, and unless otherwise stated in the context of the present invention, "-A-NH-" indicates that the amino group in the indicated CPT forms an amide bond with the carboxyl group in A.
更优选地，结构式VI-B所示化合物进一步为结构式VI-B-1所示的化合物：More preferably, the compound shown in structural formula VI-B is further a compound shown in structural formula VI-B-1:
Figure PCTCN2022139765-appb-000043
Figure PCTCN2022139765-appb-000043
或者，本发明提供的结构式VI所示的化合物进一步为结构式VI-C所示的化合物：Alternatively, the compound shown in the structural formula VI provided by the present invention is further a compound shown in the structural formula VI-C:
Figure PCTCN2022139765-appb-000044
Figure PCTCN2022139765-appb-000044
结构式VI-C中，基团A、R 8的定义与上文结构式III或结构式IIIA中基团A、R 8的定义相同。 In the structural formula VI-C, the definitions of the groups A and R 8 are the same as the definitions of the groups A and R 8 in the above structural formula III or the structural formula IIIA.
结构式VI-C中，并且在本发明的上下文中如无其他说明，“-A-NH-”表示所示氨基与A中的羧基形成酰胺键。In Formula VI-C, and unless otherwise stated in the context of the present invention, "-A-NH-" means that the indicated amino group forms an amide bond with the carboxyl group in A.
当结构式V-C中自释放结构为
Figure PCTCN2022139765-appb-000045
时，结构式V-C可进一步如结构式VI-C所示结构。 When the self-releasing structure in the structural formula VC is
Figure PCTCN2022139765-appb-000045
, the structural formula VC can be further structured as shown in the structural formula VI-C.
根据本发明的具体实施方式，在本发明的第二方面，所述化合物具有如下结构：According to a specific embodiment of the present invention, in the second aspect of the present invention, the compound has the following structure:
Figure PCTCN2022139765-appb-000046
Figure PCTCN2022139765-appb-000046
Figure PCTCN2022139765-appb-000047
Figure PCTCN2022139765-appb-000047
Figure PCTCN2022139765-appb-000048
Figure PCTCN2022139765-appb-000048
Figure PCTCN2022139765-appb-000049
Figure PCTCN2022139765-appb-000049
Figure PCTCN2022139765-appb-000050
Figure PCTCN2022139765-appb-000050
Figure PCTCN2022139765-appb-000051
Figure PCTCN2022139765-appb-000051
Figure PCTCN2022139765-appb-000052
Figure PCTCN2022139765-appb-000052
Figure PCTCN2022139765-appb-000053
Figure PCTCN2022139765-appb-000053
Figure PCTCN2022139765-appb-000054
Figure PCTCN2022139765-appb-000054
Figure PCTCN2022139765-appb-000055
Figure PCTCN2022139765-appb-000055
第三方面，本发明提供由如上结构式I、I-A、II、II-A、III、III-A、V、V-A、V-A-1、V-B、V-B-1、V-C、VI、VI-A、VI-A-1、VI-B、VI-B-1、VI-C所示化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药与抗体或抗体片段制得的抗体药物偶联物。In a third aspect, the present invention provides a structure composed of the above structural formulas I, I-A, II, II-A, III, III-A, V, V-A, V-A-1, V-B, V-B-1, V-C, VI, VI-A, VI- Antibody drug conjugates prepared from compounds shown in A-1, VI-B, VI-B-1, VI-C or pharmaceutically acceptable salts, stereoisomers, solvates or prodrugs and antibodies or antibody fragments United things.
在本发明的第三方面，所述抗体或抗体片段靶向肿瘤相关抗原，所述肿瘤相关抗原例如HER2、B7H3、HER3、CD19、CD20、CD22、CD30、CD33、CD37、CD45、CD56、 CD66e、CD70、CD74、CD73、CD79b、CD138、CD147、CD223、EpCAM、粘蛋白1(Mucin1)、STEAP1、GPNMB、FGF2、FOLR1、EGFR、EGFRvIII、组织因子(Tissuefactor)、c-MET、FGFR、Nectin 4、AGS-16、鸟苷酸环化酶C(Guanylyl cyclase C)、间皮素(Mesothelin)、SLC44A4、PSMA、EphA2、AGS-5、GPC-3、c-KIT、RoR1、PD-L1、CD27L、5T4、Mucin16、NaPi2b、STEAP、SLITRK6、ETBR、BCMA、Trop-2、CEACAM5、SC-16、SLC39A6、Delta-like protein3、Claudin 18.2。In a third aspect of the invention, the antibody or antibody fragment targets a tumor-associated antigen such as HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1 (Mucin1), STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissuefactor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C (Guanylyl cyclase C), Mesothelin (Mesothelin), SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, Claudin 18.2.
优选地，所述抗体药物偶联物具有通式
Figure PCTCN2022139765-appb-000056
所示结构，其中mAb表示抗体或抗体片段，基团M、SP 1、SP 2、A及CPT与上文结构式III或结构式IIIA中基团M、SP 1、SP 2、A及CPT的定义相同。N为1～10、优选1～8(例如1～5)、更优选3～8。 Preferably, the antibody drug conjugate has the general formula
Figure PCTCN2022139765-appb-000056
In the structure shown, wherein mAb represents an antibody or an antibody fragment, the groups M, SP 1 , SP 2 , A and CPT are the same as those defined in the above structural formula III or structural formula IIIA for the groups M, SP 1 , SP 2 , A and CPT . N is 1-10, preferably 1-8 (for example, 1-5), more preferably 3-8.
其中，EL选自如下基团，其中，
Figure PCTCN2022139765-appb-000057
表示与mAb中半胱氨酸相连，
Figure PCTCN2022139765-appb-000058
表示与M相连： Wherein, EL is selected from the following groups, wherein,
Figure PCTCN2022139765-appb-000057
Indicates that it is linked to cysteine in mAb,
Figure PCTCN2022139765-appb-000058
Indicates that it is connected to M:
E L-1a和/或E L-1b：
Figure PCTCN2022139765-appb-000059
E L-2：
Figure PCTCN2022139765-appb-000060
E L-3：
Figure PCTCN2022139765-appb-000061
E L-4：
Figure PCTCN2022139765-appb-000062
E L-5：
Figure PCTCN2022139765-appb-000063
E L-6：
Figure PCTCN2022139765-appb-000064
 EL -1a and/or EL -1b:
Figure PCTCN2022139765-appb-000059
E L -2:
Figure PCTCN2022139765-appb-000060
E L -3:
Figure PCTCN2022139765-appb-000061
E L -4:
Figure PCTCN2022139765-appb-000062
E L -5:
Figure PCTCN2022139765-appb-000063
E L -6:
Figure PCTCN2022139765-appb-000064
在所述通式结构中，mAb可以是靶向上述任何肿瘤相关抗原的IgG型抗体或其片段，优选为的IgG1亚型抗体或其片段。In the general structure, the mAb may be an IgG antibody or fragment thereof targeting any of the above tumor-associated antigens, preferably an IgG1 subtype antibody or fragment thereof.
优选地，当结构式VI所示的化合物与抗体或抗体片段偶联时，所述抗体药物偶联物具有如下通式VII或通式VIII所示的结构：Preferably, when the compound represented by the structural formula VI is coupled to an antibody or antibody fragment, the antibody-drug conjugate has a structure represented by the following general formula VII or general formula VIII:
Figure PCTCN2022139765-appb-000065
Figure PCTCN2022139765-appb-000065
其中N为1～10、优选1～8(例如1～5)、更优选3-8。Wherein N is 1-10, preferably 1-8 (eg 1-5), more preferably 3-8.
其中，Ab对应上文通式
Figure PCTCN2022139765-appb-000066
中的mAb。 Among them, Ab corresponds to the above general formula
Figure PCTCN2022139765-appb-000066
mAbs in.
通式VII和VIII中，基团A、CPT与上文第二方面中提供的结构式III或结构式IIIA中基团A、CPT的定义相同。In the general formulas VII and VIII, the definitions of the groups A and CPT are the same as the definitions of the groups A and CPT in the structural formula III or the structural formula IIIA provided in the second aspect above.
本发明在第三方面中提供的通式VII和VIII所示的抗体药物偶联物在细胞(例如肿瘤细胞)内可生成前药代谢产物。The antibody-drug conjugates represented by general formulas VII and VIII provided in the third aspect of the present invention can generate prodrug metabolites in cells (such as tumor cells).
第四方面，本发明提供结构式IX所示的化合物：In a fourth aspect, the present invention provides compounds shown in structural formula IX:
Figure PCTCN2022139765-appb-000067
Figure PCTCN2022139765-appb-000067
结构式IX中，基团A、CPT与上文第二方面中提供的结构式III或结构式IIIA中基团A、CPT的定义相同。In the formula IX, the groups A and CPT have the same definitions as the groups A and CPT in the formula III or IIIA provided in the second aspect above.
具体地，使用式VI-A化合物制备得到的抗体药物偶联物的前药代谢产物为结构式IX-A所示的化合物：Specifically, the prodrug metabolite of the antibody drug conjugate prepared using the compound of formula VI-A is the compound shown in structural formula IX-A:
Figure PCTCN2022139765-appb-000068
Figure PCTCN2022139765-appb-000068
结构式IX-A中，基团A、G、Y、R 1、R 2、R 3、R 4、X、n与上文第二方面中提供的结构式III或结构式IIIA中基团A、G、Y、R 1、R 2、R 3、R 4、X、n的定义相同。 In the structural formula IX-A, the groups A, G, Y, R 1 , R 2 , R 3 , R 4 , X, n are the same as the structural formula III provided in the second aspect above or the groups A, G, G, Y, R 1 , R 2 , R 3 , R 4 , X, and n have the same definitions.
结构式IX-A中，并且在本发明的上下文中如无其他说明，“-A-NH-”表示所示氨基与A中的羧基形成酰胺键。In Formula IX-A, and unless otherwise stated in the context of the present invention, "-A-NH-" means that the indicated amino group forms an amide bond with the carboxyl group in A.
优选地，本发明提供的结构式IX-A所示的化合物进一步为结构式IX-A-1所示的化合物：Preferably, the compound shown in the structural formula IX-A provided by the present invention is further a compound shown in the structural formula IX-A-1:
Figure PCTCN2022139765-appb-000069
Figure PCTCN2022139765-appb-000069
使用式VI-B化合物制备得到的抗体药物偶联物的前药代谢产物为结构式IX-B所示的化合物：The prodrug metabolite of the antibody drug conjugate prepared using the compound of formula VI-B is a compound shown in structural formula IX-B:
Figure PCTCN2022139765-appb-000070
Figure PCTCN2022139765-appb-000070
结构式IX-B中，基团A、G、R 5、X、n与上文第二方面中提供的结构式III或结构式IIIA中基团A、G、R 5、X、n的定义相同。 In the formula IX-B, the groups A, G, R 5 , X, n have the same definitions as the groups A, G, R 5 , X, n in the formula III or IIIA provided in the second aspect above.
结构式IX-B中，并且在本发明的上下文中如无其他说明，“-A-NH-”表示所示氨基 与A中的羧基形成酰胺键。In Formula IX-B, and unless otherwise stated in the context of the present invention, "-A-NH-" indicates that the indicated amino group forms an amide bond with the carboxyl group in A.
当使用式VI-C化合物制备得到的抗体药物偶联物的前药代谢产物为结构式IX-C所示的化合物：When the prodrug metabolite of the antibody drug conjugate prepared by using the compound of formula VI-C is the compound shown in structural formula IX-C:
Figure PCTCN2022139765-appb-000071
Figure PCTCN2022139765-appb-000071
结构式IX-C中，基团A、R8与上文第二方面中提供的结构式III或结构式IIIA中基团A、R 8的定义相同。 In the structural formula IX-C, the groups A and R8 have the same definitions as the groups A and R8 in the structural formula III or the structural formula IIIA provided in the second aspect above.
结构式IX-C中，并且在本发明的上下文中如无其他说明，“-A-NH-”表示所示氨基与A中的羧基形成酰胺键。In Formula IX-C, and unless otherwise stated in the context of the present invention, "-A-NH-" means that the indicated amino group forms an amide bond with the carboxyl group in A.
第五方面，本发明提供根据本发明的化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药或抗体药物偶联物在制备用于治疗肿瘤的药物中的用途。In the fifth aspect, the present invention provides the use of the compound according to the present invention or its pharmaceutically acceptable salts, stereoisomers, solvates or prodrugs or antibody-drug conjugates in the preparation of drugs for treating tumors.
优选地，所述肿瘤为癌症。优选地，所述肿瘤与肿瘤相关抗原例如HER2、B7H3、HER3、CD19、CD20、CD22、CD30、CD33、CD37、CD45、CD56、CD66e、CD70、CD74、CD73、CD79b、CD138、CD147、CD223、EpCAM、粘蛋白1(Mucin 1)、STEAP1、GPNMB、FGF2、FOLR1、EGFR、EGFRvIII、组织因子(Tissuefactor)、c-MET、FGFR、Nectin 4、AGS-16、鸟苷酸环化酶C(Guanylyl cyclase C)、间皮素(Mesothelin)、SLC44A4、PSMA、EphA2、AGS-5、GPC-3、c-KIT、RoR1、PD-L1、CD27L、5T4、Mucin16、NaPi2b、STEAP、SLITRK6、ETBR、BCMA、Trop-2、CEACAM5、SC-16、SLC39A6、Delta-like protein3、Claudin 18.2阳性或高表达相关。Preferably, the tumor is cancer. Preferably, the tumor and tumor-associated antigens such as HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM , Mucin 1 (Mucin 1), STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor (Tissuefactor), c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C (Guanylyl cyclase C), Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Positive or high expression of Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein 3, and Claudin 18.2.
优选地，所述肿瘤为结肠直肠癌、膀胱癌、乳腺癌、胰腺癌、肝癌、卵巢癌、子宫内膜癌、输卵管癌、胃癌、前列腺癌、小细胞肺癌、非小细胞肺癌、食道鳞状细胞癌、头颈部鳞状细胞癌、黑色素瘤、白血病、淋巴瘤、神经胶质瘤、胶质母细胞瘤。Preferably, the tumor is colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, liver cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, esophageal squamous Cell carcinoma, head and neck squamous cell carcinoma, melanoma, leukemia, lymphoma, glioma, glioblastoma.
第六方面，本发明提供采用根据本发明的化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药或抗体药物偶联物治疗肿瘤的方法，所述方法包括给有此需要的受试者施用所述化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药或抗体药物偶联物。In a sixth aspect, the present invention provides a method for treating tumors using a compound according to the present invention or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug or an antibody drug conjugate, the method comprising administering the A subject in need thereof is administered the compound or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug or antibody drug conjugate thereof.
优选地，所述肿瘤为癌症。优选地，所述肿瘤与肿瘤相关抗原例如HER2、B7H3、 HER3、CD19、CD20、CD22、CD30、CD33、CD37、CD45、CD56、CD66e、CD70、CD74、CD73、CD79b、CD138、CD147、CD223、EpCAM、粘蛋白1(Mucin 1)、STEAP1、GPNMB、FGF2、FOLR1、EGFR、EGFRvIII、组织因子(Tissuefactor)、c-MET、FGFR、Nectin 4、AGS-16、鸟苷酸环化酶C(Guanylyl cyclase C)、间皮素(Mesothelin)、SLC44A4、PSMA、EphA2、AGS-5、GPC-3、c-KIT、RoR1、PD-L1、CD27L、5T4、Mucin16、NaPi2b、STEAP、SLITRK6、ETBR、BCMA、Trop-2、CEACAM5、SC-16、SLC39A6、Delta-like protein3、Claudin 18.2阳性或高表达相关。Preferably, the tumor is cancer. Preferably, the tumor and tumor-associated antigens such as HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM , Mucin 1 (Mucin 1), STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor (Tissuefactor), c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C (Guanylyl cyclase C), Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Positive or high expression of Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein 3, and Claudin 18.2.
优选地，所述肿瘤为结肠直肠癌、膀胱癌、乳腺癌、胰腺癌、肝癌、卵巢癌、子宫内膜癌、输卵管癌、胃癌、前列腺癌、小细胞肺癌、非小细胞肺癌、食道鳞状细胞癌、头颈部鳞状细胞癌、黑色素瘤、白血病、淋巴瘤、神经胶质瘤、胶质母细胞瘤。Preferably, the tumor is colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, liver cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, esophageal squamous Cell carcinoma, head and neck squamous cell carcinoma, melanoma, leukemia, lymphoma, glioma, glioblastoma.
优选地，所述受试者为哺乳动物，优选灵长类动物，更优选为人。Preferably, the subject is a mammal, preferably a primate, more preferably a human.
附图说明Description of drawings
以下，结合附图来详细说明本发明的实施方案，其中：Below, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:
图1：本发明的抗体药物偶联物HIC分析结果。Figure 1: HIC analysis results of the antibody drug conjugate of the present invention.
图2：本发明的抗体药物偶联物在胰腺癌小鼠模型中的药效结果。Figure 2: The pharmacodynamic results of the antibody-drug conjugate of the present invention in a mouse model of pancreatic cancer.
图3：本发明的抗体药物偶联物在膀胱癌小鼠模型中的药效结果。Fig. 3: The pharmacodynamic results of the antibody-drug conjugate of the present invention in a mouse model of bladder cancer.
图4：本发明的抗体药物偶联物在肺癌小鼠模型中的药效结果。Fig. 4: The pharmacodynamic results of the antibody-drug conjugate of the present invention in a mouse model of lung cancer.
图5：本发明的抗体药物偶联物的旁观者效应研究结果(均数±SD，n＝2)。Fig. 5: Study results of the bystander effect of the antibody-drug conjugates of the present invention (mean ± SD, n = 2).
图6：本发明的抗体药物偶联物和抗体的细胞内吞时效曲线(均数±SD，n＝3)。Figure 6: Time-lapse curves of endocytosis of the antibody-drug conjugates and antibodies of the present invention (mean ± SD, n = 3).
图7：本发明的抗体药物偶联物诱导肿瘤细胞凋亡的研究结果。Fig. 7: Research results of the antibody-drug conjugates of the present invention inducing tumor cell apoptosis.
图8：本发明的抗体药物偶联物和抗体与肿瘤细胞结合的量效曲线。Fig. 8: The dose-effect curve of the binding of the antibody-drug conjugate and antibody of the present invention to tumor cells.
具体实施方式Detailed ways
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。The present invention will be described below with reference to specific examples. Those skilled in the art can understand that these examples are only for illustrating the present invention, and they do not limit the scope of the present invention in any way.
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的原料、试剂材料等，如无特殊说明，均为市售购买产品。The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials and reagent materials used in the following examples are all commercially available products unless otherwise specified.
实施例组1喜树碱类化合物的合成The synthesis of embodiment group 1 camptothecin compounds
喜树碱类化合物合成通法：Synthesis method of camptothecin compounds:
本发明的喜树碱类化合物可以由式A所示化合物与CDE三环化合物经Friedlander 反应得到，反应通式如下：The camptothecin compound of the present invention can be obtained by the Friedlander reaction of the compound shown in formula A and the CDE tricyclic compound, and the general reaction formula is as follows:
Figure PCTCN2022139765-appb-000072
Figure PCTCN2022139765-appb-000072
其中，CDE三环化合物购买自MCE：Among them, the CDE tricyclic compound was purchased from MCE:
Figure PCTCN2022139765-appb-000073
Figure PCTCN2022139765-appb-000073
HCDE三环化合物按照Bioorganic and Medicinal Chemistry，2010，vol.18，#9，p.3140-3146方法制备得到：HCDE tricyclic compounds were prepared according to Bioorganic and Medicinal Chemistry, 2010, vol.18, #9, p.3140-3146:
Figure PCTCN2022139765-appb-000074
Figure PCTCN2022139765-appb-000074
实施例组1.1式A所示化合物的合成The synthesis of compound shown in embodiment group 1.1 formula A
实施例1.1.1化合物A1的合成 The synthesis of embodiment 1.1.1 compound A1
Figure PCTCN2022139765-appb-000075
Figure PCTCN2022139765-appb-000075
方法一、按照专利公布文件WO2020200880A1方法，合成化合物A1，合成路线如下：Method 1. According to the method of patent publication WO2020200880A1, compound A1 is synthesized, and the synthetic route is as follows:
Figure PCTCN2022139765-appb-000076
Figure PCTCN2022139765-appb-000076
方法二、锌试剂钯催化偶联法合成化合物A1，合成路线如下：Method two, zinc reagent palladium catalyzed coupling method to synthesize compound A1, the synthetic route is as follows:
Figure PCTCN2022139765-appb-000077
Figure PCTCN2022139765-appb-000077
(1)乙酰化(1) Acetylation
在500mL烧瓶中加入3-溴-4-硝基苯胺(25.00g，0.12mol)和200mL醋酸，缓慢滴加50mL乙酸酐，室温下反应16小时。TLC检测原料反应完全后，将反应液过滤，反应液减压浓缩除去醋酸，合并滤饼用200mL MTBE打浆，过滤干燥的黄色固体(A1-a)27.6g。收率92％。LC-MS(ESI)：m/z 259(M+H)+。Add 3-bromo-4-nitroaniline (25.00 g, 0.12 mol) and 200 mL of acetic acid into a 500 mL flask, slowly add 50 mL of acetic anhydride dropwise, and react at room temperature for 16 hours. After the TLC detection raw material reaction is complete, the reaction solution is filtered, the reaction solution is concentrated under reduced pressure to remove acetic acid, the combined filter cake is beaten with 200mL MTBE, and 27.6g of the dried yellow solid (A1-a) is filtered. Yield 92%. LC-MS (ESI): m/z 259 (M+H)+.
(2)4-乙氧基-4-氧代丁基溴化锌的制备(2) Preparation of 4-ethoxy-4-oxobutyl zinc bromide
在一个250mL三口烧瓶中(温度计，回流冷凝管，橡胶塞)加入活化过的锌粉(19.0g，0.29mol，2.00eq)，置换氮气后加入无水DMF(145mL)，再次置换氮气，室温下加入碘单质(1.86g，0.015mol，0.1eq)，可观察到溶液由无色变为棕红色，再逐渐变为淡黄色最后恢复无色(2-3min)，然后加入4-溴丁酸乙酯(28.6g，0.15mol，1.00eq)，升温至 80℃(内温)反应4～5小时，TLC检测反应完全后，将反应液静置，冷却至室温待用，上清液呈淡黄色，浓度为1mol/L。Add activated zinc powder (19.0g, 0.29mol, 2.00eq) into a 250mL three-necked flask (thermometer, reflux condenser, rubber stopper), replace nitrogen and add anhydrous DMF (145mL), replace nitrogen again, and Add iodine simple substance (1.86g, 0.015mol, 0.1eq), the solution can be observed to change from colorless to brownish red, then gradually turn into light yellow and finally return to colorless (2-3min), then add ethyl 4-bromobutyrate Ester (28.6g, 0.15mol, 1.00eq), heat up to 80°C (internal temperature) and react for 4-5 hours. After the reaction is complete by TLC, let the reaction solution stand still and cool to room temperature for use. The supernatant is light yellow , the concentration is 1mol/L.
(3)锌试剂偶联(3) Zinc reagent coupling
500mL反应瓶中加入无水DMF(120mL)和中间体A1-a(25.0g，1.00eq)，置换氮气后加入醋酸钯(433mg，0.02eq)，置换氮气室温下搅拌10min后加入S-PHOS(1.6g，0.04eq)，再次置换氮气，搅拌20min后室温(25-30℃)滴加上述步骤中制备的4-乙氧基-4-氧代丁基溴化锌(145mL，1.50eq)，加完后保持25-30℃反应16小时。TLC检测(DCM∶EA＝5∶1)原料反应完全后，将反应液冷却至室温，向反应液中加入氯化铵溶液(15mL)淬灭反应。将反应液倒入1L水中，加入400mL乙酸乙酯，分液，布氏漏斗过滤，水相用乙酸乙酯(400mL*2)萃取，有机相用水(500mL)洗涤两遍，饱和氯化钠溶液(500mL)洗涤一遍，无水硫酸钠干燥，减压浓缩，得红棕色油状液体(A1-b)37.0g，收率130％。粗产品不用进一步纯化，直接用于下一步反应。LC-MS(ESI)：[M+1]+＝295。Anhydrous DMF (120mL) and intermediate A1-a (25.0g, 1.00eq) were added to a 500mL reaction flask, palladium acetate (433mg, 0.02eq) was added after nitrogen replacement, and S-PHOS ( 1.6g, 0.04eq), nitrogen was replaced again, and after stirring for 20min, 4-ethoxy-4-oxobutylzinc bromide (145mL, 1.50eq) prepared in the above step was added dropwise at room temperature (25-30°C), After the addition, keep the reaction at 25-30°C for 16 hours. TLC detection (DCM:EA=5:1) After the reaction of the raw materials was complete, the reaction solution was cooled to room temperature, and ammonium chloride solution (15 mL) was added to the reaction solution to quench the reaction. Pour the reaction solution into 1L of water, add 400mL of ethyl acetate, separate the layers, filter through a Buchner funnel, extract the aqueous phase with ethyl acetate (400mL*2), wash the organic phase twice with water (500mL), and wash with saturated sodium chloride solution (500 mL) was washed once, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 37.0 g of reddish-brown oily liquid (A1-b) with a yield of 130%. The crude product was directly used in the next reaction without further purification. LC-MS (ESI): [M+1]+=295.
(4)中间体A1-c的合成(4) Synthesis of Intermediate A1-c
3L三口反应瓶中加入中间体A1-b(37.0g，1.0eq)和乙醇(1.5L)，原料完全溶解。反应体系冷却至5℃，加入5％Pd/C(5.7g，1.0eq)，氢气氛围下(常压)升温至25℃反应16小时。TLC检测(DCM∶EA＝5∶1)反应完毕，反应体系在硅藻土下过滤，有机相浓缩得粗产品(A1-c)33.0g，收率130％。粗产品不用进一步纯化，直接用于下一步反应。LC-MS(ESI)：[M+1]+＝265。Intermediate A1-b (37.0 g, 1.0 eq) and ethanol (1.5 L) were added to a 3 L three-neck reaction flask, and the raw materials were completely dissolved. The reaction system was cooled to 5° C., 5% Pd/C (5.7 g, 1.0 eq) was added, and the temperature was raised to 25° C. under a hydrogen atmosphere (normal pressure) to react for 16 hours. TLC detected (DCM:EA=5:1) that the reaction was complete, the reaction system was filtered under celite, and the organic phase was concentrated to obtain 33.0 g of crude product (A1-c), with a yield of 130%. The crude product was directly used in the next reaction without further purification. LC-MS (ESI): [M+1]+=265.
(5)中间体A1-d的合成(5) Synthesis of Intermediate A1-d
在1L烧瓶中加入中间体A1-c(33.0g，1.00eq)和260mL醋酸，缓慢滴加46mL乙酸酐，室温下反应2小时。TLC检测(DCM∶MeOH＝10∶1)原料反应完全后，将反应液过滤，反应液减压浓缩除去醋酸，加入250mL水，水相用乙酸乙酯(400mL*2)萃取，有机相用水(500mL)洗涤两遍，饱和氯化钠溶液(500mL)洗涤一遍，无水硫酸钠干燥，减压浓缩，得粗产品32g。粗产品用400mL MTBE打浆，过滤干燥的淡黄色固体(A1-d)27.0g，收率91％。四步反应总收率为83.7％。Add intermediate A1-c (33.0 g, 1.00 eq) and 260 mL of acetic acid into a 1 L flask, slowly add 46 mL of acetic anhydride dropwise, and react at room temperature for 2 hours. TLC detection (DCM: MeOH = 10: 1) After the raw materials have reacted completely, the reaction solution is filtered, the reaction solution is concentrated under reduced pressure to remove acetic acid, 250 mL of water is added, the aqueous phase is extracted with ethyl acetate (400 mL*2), and the organic phase is extracted with water ( 500 mL) and washed twice with saturated sodium chloride solution (500 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 32 g of a crude product. The crude product was slurried with 400mL MTBE, and 27.0g of the light yellow solid (A1-d) was filtered and dried, with a yield of 91%. The total yield of the four-step reaction is 83.7%.
LC-MS(ESI)：[M+1]+＝307。LC-MS (ESI): [M+1]+=307.
1H NMR(400MHz，DMSO)δ9.87(s，1H)，9.19(s，1H)，7.41(s，1H)，7.37(d，J＝8.7Hz，1H)，7.20(d，J＝8.5Hz，1H)，4.06(q，J＝7.0Hz，2H)，2.52(d，J＝8.5Hz，2H)，2.29(t，J＝7.3Hz，2H)，2.02(s，6H)，1.79-1.64(m，2H)，1.18(t，J＝7.1Hz，3H).1H NMR (400MHz, DMSO) δ9.87(s, 1H), 9.19(s, 1H), 7.41(s, 1H), 7.37(d, J=8.7Hz, 1H), 7.20(d, J=8.5Hz , 1H), 4.06(q, J=7.0Hz, 2H), 2.52(d, J=8.5Hz, 2H), 2.29(t, J=7.3Hz, 2H), 2.02(s, 6H), 1.79-1.64 (m, 2H), 1.18 (t, J=7.1Hz, 3H).
(6)中间体A1-e的合成(6) Synthesis of Intermediate A1-e
500mL三口反应瓶中加入中间体A1-d(27.0g，88mmol，1.0eq)，水(100mL)和四氢呋喃(200mL)，原料完全溶解。室温条件下加入一水合氢氧化锂(18.5g，441mmol， 5.0eq)，反应体系维持室温继续反应3小时。TLC检测反应完毕，减压蒸出大部分四氢呋喃，残留物加入300mL水，水相用EA萃取(2×100mL)，弃去有机相。水相在冰浴下用6N盐酸调pH至4，有固体析出，过滤得白色固体(中间体A1-e)，19.1g，收率78％。Intermediate A1-d (27.0g, 88mmol, 1.0eq), water (100mL) and tetrahydrofuran (200mL) were added to a 500mL three-neck reaction flask, and the raw materials were completely dissolved. Lithium hydroxide monohydrate (18.5 g, 441 mmol, 5.0 eq) was added at room temperature, and the reaction system was maintained at room temperature for 3 hours. The completion of the reaction was detected by TLC, most of the tetrahydrofuran was distilled off under reduced pressure, the residue was added to 300 mL of water, the aqueous phase was extracted with EA (2×100 mL), and the organic phase was discarded. The pH of the aqueous phase was adjusted to 4 with 6N hydrochloric acid in an ice bath, and a solid precipitated out, which was filtered to obtain a white solid (Intermediate A1-e), 19.1 g, with a yield of 78%.
1H NMR(400MHz，DMSO)δ12.09(s，1H)，9.86(s，1H)，9.18(s，1H)，7.38(d，J＝15.5Hz，2H)，7.23(d，J＝8.5Hz，1H)，2.51(d，J＝8.1Hz，2H)，2.23(t，J＝7.1Hz，2H)，2.02(s，6H)，1.76-1.61(m，2H)1H NMR (400MHz, DMSO) δ12.09(s, 1H), 9.86(s, 1H), 9.18(s, 1H), 7.38(d, J=15.5Hz, 2H), 7.23(d, J=8.5Hz , 1H), 2.51(d, J=8.1Hz, 2H), 2.23(t, J=7.1Hz, 2H), 2.02(s, 6H), 1.76-1.61(m, 2H)
(7)化合物A1的合成(7) Synthesis of Compound A1
250mL反应瓶中加入多聚磷酸40ml，加热至90℃，分批加入中间体A1-5(5.0g，17.97mmol)，内温保持95-100℃反应5小时。TLC检测反应完毕，撤去加热，体系降温至50-60℃，向反应体系中滴加15mL 4M HCl(aq)(温度会升至100℃)淬灭反应，滴毕，冰浴下滴加600mL 4M NaOH水溶剂调pH到10。水相用乙酸乙酯(50mL*3)萃取，合并有机相，用饱和氯化钠溶液(50mL)洗涤一遍，无水硫酸钠干燥，减压浓缩，得黄色固体(中间体A1-f)3.75g，收率80.5％。不经纯化直接下一步反应。LC-MS(ESI)：[M+1]+＝261。Add 40ml of polyphosphoric acid into a 250mL reaction bottle, heat to 90°C, add intermediate A1-5 (5.0g, 17.97mmol) in batches, and keep the internal temperature at 95-100°C for 5 hours. TLC detects that the reaction is complete, remove the heat, cool the system to 50-60°C, add 15mL 4M HCl(aq) dropwise to the reaction system (the temperature will rise to 100°C) to quench the reaction, after the drop is complete, add 600mL 4M HCl(aq) dropwise in an ice bath NaOH aqueous solution to adjust the pH to 10. The aqueous phase was extracted with ethyl acetate (50 mL*3), the organic phases were combined, washed once with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a yellow solid (Intermediate A1-f) 3.75 g, yield 80.5%. The next reaction was carried out directly without purification. LC-MS (ESI): [M+1]+=261.
250mL三口反应瓶中将原料中间体A1-f(3.75g)悬浮于19％盐酸(30mL)中。反应体系加热至90℃(内温)反应3小时。TLC检测原料反应完毕，冰盐浴降温至5℃以下，滴加4M NaOH溶液(45mL，30eq)调pH到10，水相用乙酸乙酯(80mL*5)萃取，合并有机相，用饱和氯化钠溶液(50mL)洗涤一遍，无水硫酸钠干燥，减压浓缩，得粗产品2.45g。粗产品柱层析纯化，洗脱剂为DCM，得黄色固体(化合物A1)，1.93g，收率76％。两步反应总收率61.2％。In a 250 mL three-necked reaction flask, the raw material intermediate A1-f (3.75 g) was suspended in 19% hydrochloric acid (30 mL). The reaction system was heated to 90° C. (internal temperature) for 3 hours. The reaction of the raw materials was detected by TLC, the temperature of the ice-salt bath was lowered to below 5°C, and 4M NaOH solution (45mL, 30eq) was added dropwise to adjust the pH to 10. Wash once with sodium chloride solution (50 mL), dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 2.45 g of a crude product. The crude product was purified by column chromatography with DCM as the eluent to obtain a yellow solid (Compound A1), 1.93 g, with a yield of 76%. The total yield of the two-step reaction is 61.2%.
LC-MS(ESI)：[M+1]+＝177。LC-MS (ESI): [M+1]+=177.
1H NMR(400MHz，DMSO)δ6.76(d，J＝8.7Hz，1H)，6.68(s，2H)，6.42(d，J＝8.7Hz，1H)，4.17(s，2H)，2.55(t，J＝5.9Hz，2H)，2.46(t，J＝6.2Hz，2H)，2.00-1.80(m，2H)1H NMR (400MHz, DMSO) δ6.76(d, J=8.7Hz, 1H), 6.68(s, 2H), 6.42(d, J=8.7Hz, 1H), 4.17(s, 2H), 2.55(t , J=5.9Hz, 2H), 2.46(t, J=6.2Hz, 2H), 2.00-1.80(m, 2H)
方法三、内酰胺水解法制备化合物A1，合成路线如下：Method 3: compound A1 is prepared by lactam hydrolysis, and the synthetic route is as follows:
Figure PCTCN2022139765-appb-000078
Figure PCTCN2022139765-appb-000078
根据专利公布文件CN106349233A方法制备得到氨基内酰胺化合物。The aminolactam compound is prepared according to the method of the patent publication CN106349233A.
Figure PCTCN2022139765-appb-000079
Figure PCTCN2022139765-appb-000079
于500ml三口烧瓶中，将氨基内酰胺化合物(17.6g，0.1moml)，乙醇(250ml)，98％硫酸(5ml)混合，加热回流反应24小时。取样检测，待反应完全后，将反应液减压浓缩至干。残留物加入二氯甲烷(200ml)和水(100ml)，冰浴降温至10℃以下，用1N氢氧化钠水溶液调节pH7～8，搅拌分液，水相用二氯甲烷萃取，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，抽滤，减压蒸馏，蒸除有机溶剂，得到二氨基乙酯中间体粗品，25g，直接下一步反应。In a 500ml three-neck flask, the aminolactam compound (17.6g, 0.1moml), ethanol (250ml), and 98% sulfuric acid (5ml) were mixed, and heated to reflux for 24 hours. Sampling was performed for testing. After the reaction was complete, the reaction solution was concentrated to dryness under reduced pressure. Dichloromethane (200ml) and water (100ml) were added to the residue, cooled to below 10°C in an ice bath, adjusted to pH 7-8 with 1N aqueous sodium hydroxide solution, stirred and separated, the aqueous phase was extracted with dichloromethane, and the organic phases were combined. Wash with saturated brine, dry over anhydrous sodium sulfate, filter with suction, and distill under reduced pressure to remove the organic solvent to obtain 25 g of the crude diaminoethyl ester intermediate, which is directly used for the next reaction.
二氨基乙酯中间体溶于二氯甲烷(200ml)中，加入三乙胺(20.2g，0.2mol，2eq)，冰浴降温至10℃以下，滴加乙酸酐(25.5g，0.25mol，2.5eq)。滴毕，保温反应1小时，取样检测，待反应完全后，将反应液倒入冰的1N盐酸中，搅拌15分钟，分液，水相二氯甲烷(50ml*3)萃取，合并有机相，无水硫酸钠干燥，抽滤，减压蒸馏，蒸除有机溶剂，粗产品用400mL MTBE打浆，过滤干燥得中间体A1-e为淡黄色固体，26.9g。两步反应总收率87.8％。LC-MS(ESI)：m/z 307(M+H)+。The diaminoethyl ester intermediate was dissolved in dichloromethane (200ml), triethylamine (20.2g, 0.2mol, 2eq) was added, the temperature was cooled to below 10°C in an ice bath, and acetic anhydride (25.5g, 0.25mol, 2.5 eq). After dropping, keep warm for 1 hour, take a sample for detection, after the reaction is complete, pour the reaction solution into iced 1N hydrochloric acid, stir for 15 minutes, separate the liquids, extract the aqueous phase with dichloromethane (50ml*3), combine the organic phases, Dry over anhydrous sodium sulfate, filter with suction, distill under reduced pressure to remove the organic solvent, beat the crude product with 400mL MTBE, filter and dry to obtain intermediate A1-e as light yellow solid, 26.9g. The total yield of the two-step reaction is 87.8%. LC-MS (ESI): m/z 307 (M+H)+.
再根据方法2，从中间体A1-e制备得到化合物A1。Then according to method 2, compound A1 was prepared from intermediate A1-e.
实施例1.1.2化合物A2的合成 The synthesis of embodiment 1.1.2 compound A2
Figure PCTCN2022139765-appb-000080
Figure PCTCN2022139765-appb-000080
按照专利公布文件WO2021148501方法，合成化合物A2，合成路线如下：According to the method of patent publication WO2021148501, compound A2 was synthesized, and the synthetic route is as follows:
Figure PCTCN2022139765-appb-000081
Figure PCTCN2022139765-appb-000081
实施例1.1.3化合物A3的合成 The synthesis of embodiment 1.1.3 compound A3
Figure PCTCN2022139765-appb-000082
Figure PCTCN2022139765-appb-000082
按照专利公布文件US2004266803A方法，合成化合物A3。Compound A3 was synthesized according to the method of patent publication US2004266803A.
实施例1.1.4化合物A4的合成 The synthesis of embodiment 1.1.4 compound A4
按照与文献Journal of Medicinal chemistry，1998，41(13)，2308-2318类似的方法合成化合物A4，合成路线如下：Compound A4 was synthesized according to the method similar to the literature Journal of Medicinal chemistry, 1998, 41 (13), 2308-2318, and the synthetic route is as follows:
Figure PCTCN2022139765-appb-000083
Figure PCTCN2022139765-appb-000083
步骤1：step 1:
A4-2中间体由A4-1化合物按照文献方法制备得到。The A4-2 intermediate was prepared from the A4-1 compound according to the literature method.
步骤2：Step 2:
于-5～5℃下，将A4-2中间体(3.4g)溶于二氯甲烷中，加入三乙胺(3.0g)，继续慢慢滴加AllocCl(2.8g)，滴加完毕搅拌反应1～2小时；加水淬灭，水洗一次，饱和食盐水洗涤一次，无水硫酸钠干燥，浓缩干得A4-3中间体粗品5.5g。LC-MS(ESI)：[M+1]+＝255.7。Dissolve the A4-2 intermediate (3.4g) in dichloromethane at -5~5°C, add triethylamine (3.0g), continue to drop AllocCl (2.8g) slowly, and stir the reaction after the addition is complete 1 to 2 hours; add water to quench, wash once with water, wash once with saturated brine, dry over anhydrous sodium sulfate, and concentrate to dryness to obtain 5.5 g of crude intermediate A4-3. LC-MS (ESI): [M+1]+=255.7.
步骤3：Step 3:
常温下，将A4-3中间体(5.5g)混于TBAF(55ml)和丙烯酸(110ml)中，加热至50～55℃搅拌反应24小时；反应也直接浓缩干，柱层析，石油醚洗脱至甲醇∶二氯甲烷＝1∶ 50得A4-4中间体粗品约5.2g。LC-MS(ESI)：[M+1]+＝327.4。At room temperature, the A4-3 intermediate (5.5g) was mixed with TBAF (55ml) and acrylic acid (110ml), heated to 50-55°C and stirred for 24 hours; the reaction was also directly concentrated to dryness, column chromatography, petroleum ether washing Removal to methanol: dichloromethane = 1: 50 yielded about 5.2 g of crude intermediate A4-4. LC-MS (ESI): [M+1]+=327.4.
步骤4：Step 4:
常温下，将A4-4中间体(5.0g)混于乙醇∶水＝3∶1的100ml混合溶液中，加入氯化铵(5.5g)和铁粉(5.5g)，加热至回流反应1～2小时；反应也冷却至室温，过滤，固体用乙醇洗涤，浓缩干，得A4-5中间体粗品约3.8g，LC-MS(ESI)：[M+1]+＝297.4。At room temperature, mix the A4-4 intermediate (5.0g) in a 100ml mixed solution of ethanol: water = 3:1, add ammonium chloride (5.5g) and iron powder (5.5g), and heat to reflux for 1- 2 hours; the reaction was also cooled to room temperature, filtered, the solid was washed with ethanol, and concentrated to dryness to obtain about 3.8 g of crude intermediate A4-5, LC-MS (ESI): [M+1]+=297.4.
步骤5：Step 5:
20～30℃，将A4-5中间体(3.8g)混于三氟乙酸(38ml)中，加入三氟乙酸酐(38ml)，搅拌反应18～24小时；后处理：直接浓缩干，柱层析，石油醚洗脱至乙酸乙酯∶石油醚＝1∶4得A4-6中间体：1.0g；LC-MS(ESI)：[M+1]+＝375.3。20～30℃, mix A4-5 intermediate (3.8g) in trifluoroacetic acid (38ml), add trifluoroacetic anhydride (38ml), stir and react for 18～24 hours; Analysis, petroleum ether was eluted to ethyl acetate: petroleum ether = 1: 4 to obtain A4-6 intermediate: 1.0 g; LC-MS (ESI): [M+1]+ = 375.3.
步骤6：Step 6:
常温下，将A4-6中间体(1.0g)混于甲醇(20ml)中，加入碳酸钾(2.0g)和水(5ml)搅拌反应1～2小时；加水稀释反应液，乙酸乙酯萃取，有机相饱和食盐水洗涤，无水硫酸钠干燥，浓缩干得粗品，柱层析石油醚洗脱至乙酸乙酯∶石油醚＝1∶2得A4-7中间体：0.65g，LC-MS(ESI)：[M+1]+＝279.3。At room temperature, mix A4-6 intermediate (1.0g) in methanol (20ml), add potassium carbonate (2.0g) and water (5ml) and stir for 1-2 hours; add water to dilute the reaction solution, extract with ethyl acetate, The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain a crude product. Column chromatography was eluted with petroleum ether until ethyl acetate: petroleum ether = 1:2 to obtain intermediate A4-7: 0.65 g, LC-MS ( ESI): [M+1]+=279.3.
步骤7：Step 7:
常温下，将A4-7中间体(500mg)溶于四氢呋喃(20ml)中，氮气保护下，加入吡咯(120mg)和四三苯基膦钯(160mg)，完毕搅拌反应1～2小时；将反应液直接浓缩干，柱层析二氯甲烷∶甲醇＝30∶1纯化，得到A4为棕色固体，30mg；LC-MS(ESI)：[M+1]+＝195.4。At room temperature, the A4-7 intermediate (500mg) was dissolved in tetrahydrofuran (20ml), and under nitrogen protection, pyrrole (120mg) and tetrakistriphenylphosphine palladium (160mg) were added, and the stirring reaction was completed for 1 to 2 hours; The solution was directly concentrated to dryness and purified by column chromatography with dichloromethane:methanol=30:1 to obtain A4 as a brown solid, 30 mg; LC-MS (ESI): [M+1]+=195.4.
实施例1.1.5化合物A5的合成 The synthesis of embodiment 1.1.5 compound A5
按照与文献Journal of Medicinal chemistry，1998，41(13)，2308-2318类似的方法合成化合物A5，合成路线如下：Compound A5 was synthesized according to a method similar to the literature Journal of Medicinal chemistry, 1998, 41 (13), 2308-2318, and the synthetic route is as follows:
Figure PCTCN2022139765-appb-000084
Figure PCTCN2022139765-appb-000084
步骤1：step 1:
20～30℃，将A5-1化合物(25g)混于乙酸(100ml)中，慢慢加入乙酸酐(24.9g)， 加料完毕，搅拌3～4小时；将反应液慢慢转入冰水中，搅拌析出固体，过滤，收集固体，水洗，真空干燥得A5-2中间体为黄色固体，31.0g，LC-MS(ESI)：[M+1]+＝197.2。20-30°C, mix A5-1 compound (25g) in acetic acid (100ml), slowly add acetic anhydride (24.9g), after the addition is complete, stir for 3-4 hours; slowly transfer the reaction solution into ice water, Stir to precipitate a solid, filter, collect the solid, wash with water, and dry in vacuo to obtain intermediate A5-2 as a yellow solid, 31.0 g, LC-MS (ESI): [M+1]+=197.2.
步骤2：Step 2:
20～30℃，将A5-2中间体(29g)、碳酸钾(40g)、碘化钾(5g)和溴丙醇(25g)混于DMF(300ml)中，加热至100～110℃，搅拌3～4小时；反应液冷却至室温，加入冰水中淬灭，用乙酸乙酯2L萃取三次，合并，饱和食盐水洗涤，无水硫酸钠干燥，浓缩干用石油醚打浆得固体，真空干燥得A5-3中间体，为黄色固体：33.0g，LC-MS(ESI)：[M+1]+＝255.3。20～30℃, mix A5-2 intermediate (29g), potassium carbonate (40g), potassium iodide (5g) and bromopropanol (25g) in DMF (300ml), heat to 100～110℃, stir for 3～ 4 hours; the reaction solution was cooled to room temperature, quenched by adding ice water, extracted three times with 2 L of ethyl acetate, combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated to dryness and beaten with petroleum ether to obtain a solid, which was dried in vacuo to obtain A5- 3 intermediate as a yellow solid: 33.0 g, LC-MS (ESI): [M+1]+=255.3.
步骤3：Step 3:
20～25℃，将A5-3中间体(23g)溶于乙腈(1L)中，依次加入磷酸二氢钠溶液(0.67mol，pH 6.7，900ml)、TEMPO(5g)和亚氯酸钠溶液(52.0g溶于60ml水中)和次氯酸钠溶液(38ml混合38ml水)，加料完毕，反应液呈现深棕色，搅拌30分钟；将反应液冷却至室温，用2N的盐酸调节pH至2～3，乙酸乙酯萃取(1000ml×3)，乙酸乙酯层合并，饱和食盐水洗涤，无水硫酸钠干燥，过滤浓缩干，石油醚打浆得A5-4中间体粗品24.0g，LC-MS(ESI)：[M+1]+＝269.2。20～25 ℃, the A5-3 intermediate (23g) was dissolved in acetonitrile (1L), and sequentially added sodium dihydrogen phosphate solution (0.67mol, pH 6.7, 900ml), TEMPO (5g) and sodium chlorite solution ( 52.0g dissolved in 60ml water) and sodium hypochlorite solution (38ml mixed with 38ml water), after the addition, the reaction solution was dark brown and stirred for 30 minutes; Ester extraction (1000ml×3), ethyl acetate layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to dryness, beaten with petroleum ether to obtain 24.0g of crude intermediate A5-4, LC-MS (ESI): [ M+1]+=269.2.
步骤4：Step 4:
常温下，将A5-4中间体(25g)和5％钯炭(5g)混于甲醇(500ml)中，加压氢化反应3～4小时；过滤掉钯炭，固体用甲醇洗涤，滤液浓缩，残留物石油醚打浆得粗品，真空干燥得A5-5中间体，为黄色固体：18.0g，LC-MS(ESI)：[M+1]+＝239.5。At room temperature, the A5-4 intermediate (25g) and 5% palladium carbon (5g) were mixed in methanol (500ml), and hydrogenated under pressure for 3 to 4 hours; the palladium carbon was filtered off, the solid was washed with methanol, and the filtrate was concentrated. The residue was beaten with petroleum ether to obtain a crude product, which was dried in vacuo to obtain intermediate A5-5 as a yellow solid: 18.0 g, LC-MS (ESI): [M+1]+=239.5.
按照化合物A4合成中步骤5，步骤6类似的方法，从A5-5中间体出发合成A5-7中间体，最后用6N盐酸脱除乙酰基保护，制备得到化合物A5，为黄色固体，LC-MS(ESI)：[M+1]+＝179.2。According to the method similar to step 5 and step 6 in the synthesis of compound A4, the intermediate A5-7 was synthesized from the intermediate A5-5, and finally the acetyl group was deprotected with 6N hydrochloric acid to prepare compound A5 as a yellow solid, LC-MS (ESI): [M+1]+=179.2.
实施例1.1.6化合物A6的合成 The synthesis of embodiment 1.1.6 compound A6
按照文献Journal ofMedicinal chemistry，1998，41(13)，2308-2318方法合成化合物A6，合成路线如下：Synthesize compound A6 according to the literature Journal of Medicinal chemistry, 1998, 41 (13), 2308-2318 method, synthetic route is as follows:
Figure PCTCN2022139765-appb-000085
Figure PCTCN2022139765-appb-000085
化合物A6为黄色固体，LC-MS(ESI)：[M+1]+＝248.9。Compound A6 is a yellow solid, LC-MS (ESI): [M+1]+=248.9.
1H NMR(400MHz，d6-DMSO)：δ12.289(1H，s)，8.666-8.648(1H，d)，8.198-8.1(1H，d)，3.138-3.108(2H，t)，2.768-2.734(2H，t)，2.219(3H，s)，2.050-1.991(2H，m) 1 H NMR (400MHz, d6-DMSO): δ12.289 (1H, s), 8.666-8.648 (1H, d), 8.198-8.1 (1H, d), 3.138-3.108 (2H, t), 2.768-2.734 (2H,t), 2.219(3H,s), 2.050-1.991(2H,m)
步骤1：step 1:
于250ml三口烧瓶中，氮气保护下，将A1-4化合物(1.25g，5mmol，1eq)溶于150ml四氢呋喃中，降温到-60℃，加入LDA(2M in THF，7.6ml，15.2mmol，3.04eq)，加毕后于-60℃下搅拌反应30分钟。加入MeI(1.45g，10.21mmol，2.04eq)，加毕，撤除干冰浴，自然升温，室温反应过夜。加入50ml氯化铵水溶液淬灭反应，用乙酸乙酯(150ml*3)萃取，合并有机相，用水洗涤一次，有机相浓缩至干得粗品。粗品经柱层析纯化，洗脱剂为石油醚∶乙酸乙酯＝1∶0～5∶1，得A6-1化合物，黄色固体，350mg，收率28％。In a 250ml three-necked flask, under the protection of nitrogen, the A1-4 compound (1.25g, 5mmol, 1eq) was dissolved in 150ml of tetrahydrofuran, cooled to -60°C, added LDA (2M in THF, 7.6ml, 15.2mmol, 3.04eq ), after the addition was completed, the reaction was stirred at -60°C for 30 minutes. MeI (1.45g, 10.21mmol, 2.04eq) was added, after the addition was complete, the dry ice bath was removed, the temperature was raised naturally, and the reaction was carried out overnight at room temperature. Add 50ml of ammonium chloride aqueous solution to quench the reaction, extract with ethyl acetate (150ml*3), combine the organic phases, wash with water once, and concentrate the organic phases to dryness to obtain a crude product. The crude product was purified by column chromatography, and the eluent was petroleum ether: ethyl acetate = 1:0-5:1 to obtain compound A6-1 as a yellow solid, 350 mg, with a yield of 28%.
1H NMR(CDCl 3)：δ12.49(1H，s)，8.77(1H，d)，8.03(1H，d)，3.21(2H，m)，2.22(3H，s)，1.90(2H，t)，1.20(6H，s) 1 H NMR (CDCl 3 ): δ12.49 (1H, s), 8.77 (1H, d), 8.03 (1H, d), 3.21 (2H, m), 2.22 (3H, s), 1.90 (2H, t ), 1.20(6H,s)
步骤2：Step 2:
于100ml单口烧瓶中，将A6-1化合物(280mg)与20ml 6N盐酸混合，加热回流搅拌反应2小时；停止加热，待反应液冷却至室温后，用碳酸氢钠将反应液pH调到7～8，用二氯甲烷(30ml*3)萃取，合并有机相，浓缩干，得A6-2化合物，棕灰色固体，275mg。粗品不经纯化，直接下一步反应。In a 100ml single-necked flask, mix A6-1 compound (280mg) with 20ml 6N hydrochloric acid, heat and reflux and stir for 2 hours; stop heating, and after the reaction solution is cooled to room temperature, use sodium bicarbonate to adjust the pH of the reaction solution to 7~ 8. Extract with dichloromethane (30ml*3), combine the organic phases, and concentrate to dryness to obtain compound A6-2 as a brown-gray solid, 275mg. The crude product was directly reacted in the next step without purification.
步骤3：Step 3:
于50ml三口烧瓶中，将A6-2化合物(220mg)溶于乙酸(25ml)中，加入1.3克铁粉，加毕，于80～85℃搅拌1-2小时；待反应完全后，减压蒸馏蒸除乙酸，残留物加入水(30ml)，用碳酸氢钠将pH调到7～8，用二氯甲烷(30ml*3)萃取，浓缩干得粗品，粗品经柱层析纯化，洗脱剂为石油醚∶乙酸乙酯＝1∶0～2∶1，得A6化合物，棕色固体，122mg，两步反应收率75％。LC-MS(ESI)：[M+1]+＝205.0In a 50ml three-neck flask, dissolve compound A6-2 (220mg) in acetic acid (25ml), add 1.3g of iron powder, after the addition is complete, stir at 80-85°C for 1-2 hours; after the reaction is complete, distill under reduced pressure Evaporate the acetic acid, add water (30ml) to the residue, adjust the pH to 7-8 with sodium bicarbonate, extract with dichloromethane (30ml*3), concentrate and dry to obtain the crude product, which is purified by column chromatography, eluent Petroleum ether: ethyl acetate = 1:0 ~ 2:1 to obtain compound A6 as a brown solid, 122 mg, and the yield of the two-step reaction was 75%. LC-MS (ESI): [M+1]+=205.0
实施例1.1.7化合物A7的合成 Synthesis of Example 1.1.7 Compound A7
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000086
Figure PCTCN2022139765-appb-000086
于250ml三口烧瓶中，将A1化合物(2.18g，124mmol，1eq)溶于四氢呋喃(100ml)中，加入Boc酸酐(8.2g，376mmol，3eq)，于40～45℃搅拌反应5小时；将反应液直接浓缩干，经柱层析纯化，洗脱剂为石油醚∶乙酸乙酯＝1∶0～1∶3，得A7-1化合物，黄色固体3.0g，收率90％。In a 250ml three-necked flask, the A1 compound (2.18g, 124mmol, 1eq) was dissolved in tetrahydrofuran (100ml), and Boc anhydride (8.2g, 376mmol, 3eq) was added, and the reaction was stirred at 40-45°C for 5 hours; Directly concentrated to dryness, purified by column chromatography, the eluent is petroleum ether: ethyl acetate = 1:0 ~ 1:3, to obtain compound A7-1, 3.0 g of yellow solid, yield 90%.
1H NMR(400MHz，CDCl3)δ7.25(s，1H)，6.41(t，J＝10.2Hz，3H)，5.90(s，1H)，2.73(t，J＝6.2Hz，2H)，2.61-2.47(m，2H)，2.02-1.86(m，3H)，1.49-1.36(m，9H)1H NMR (400MHz, CDCl3) δ7.25(s, 1H), 6.41(t, J=10.2Hz, 3H), 5.90(s, 1H), 2.73(t, J=6.2Hz, 2H), 2.61-2.47 (m, 2H), 2.02-1.86 (m, 3H), 1.49-1.36 (m, 9H)
于250ml三口烧瓶中，将A7-1化合物(3.0g，10.8mmol，1eq)和DIPEA(3.5g，27.1mmol，1.5eq)混于二氯甲烷(125ml)中，用冰盐浴将反应液冷却至-5～0℃，滴加乙酰氯(1.3g，16.5mmol，2eq)，滴毕，撤除冰盐浴，自然升温，于室温下搅拌反应4小时；将反应液浓缩干，残留物经柱层析纯化，洗脱剂为石油醚∶乙酸乙酯＝1∶0～1∶3，得A7-2化合物，淡黄色固体，3.48g，收率100％。In a 250ml three-necked flask, A7-1 compound (3.0g, 10.8mmol, 1eq) and DIPEA (3.5g, 27.1mmol, 1.5eq) were mixed in methylene chloride (125ml), and the reaction solution was cooled with an ice-salt bath To -5 ~ 0 ℃, dropwise add acetyl chloride (1.3g, 16.5mmol, 2eq), dropwise, remove the ice-salt bath, raise the temperature naturally, stir and react at room temperature for 4 hours; Purified by chromatography, the eluent was petroleum ether: ethyl acetate = 1:0 ~ 1:3, to obtain compound A7-2, light yellow solid, 3.48g, yield 100%.
1H NMR(400MHz，CDCl3)δ12.01(s，1H)，8.55(d，J＝9.1Hz，1H)，7.57(t，J＝36.2Hz，1H)，6.09(s，1H)，2.80(t，J＝6.2Hz，2H)，2.65-2.58(m，2H)，2.15(s，3H)，2.07-1.98(m，2H)，1.44(s，9H)1H NMR (400MHz, CDCl3) δ12.01(s, 1H), 8.55(d, J=9.1Hz, 1H), 7.57(t, J=36.2Hz, 1H), 6.09(s, 1H), 2.80(t , J=6.2Hz, 2H), 2.65-2.58(m, 2H), 2.15(s, 3H), 2.07-1.98(m, 2H), 1.44(s, 9H)
于500ml三口烧瓶中，氮气保护下，将A7-2化合物(2.45g，7.7mmol，1eq)溶于四氢呋喃(200ml)中，用干冰-丙酮浴将反应液冷却至-70～-60℃，慢慢滴加KHMDS(1M，31ml，31.0mmol，4eq)，滴毕，于-70～-60℃搅拌10分钟；滴加NFSI的四氢呋喃溶液(将NFSI(7.35g，23mmol，3eq)溶于四氢呋喃(70ml))，滴毕，于-70～-60℃搅拌10分钟，自然升温至20～30℃并搅3小时；加入饱和氯化铵水溶液(80ml)淬灭反应，反应液用乙酸乙酯(150ml*3)萃取，合并有机相，饱和食盐水洗涤，浓缩干得粗品，经柱层析纯化，洗脱剂为石油醚∶乙酸乙酯＝1∶0-1∶3，得A7-3化合物，黄色固体，0.85g，收率31.2％。LC-MS(ESI)：[M+1]+＝355.8In a 500ml three-neck flask, under the protection of nitrogen, the A7-2 compound (2.45g, 7.7mmol, 1eq) was dissolved in tetrahydrofuran (200ml), and the reaction solution was cooled to -70～-60°C with a dry ice-acetone bath, and slowly Slowly add KHMDS (1M, 31ml, 31.0mmol, 4eq) dropwise, after dropping, stir at -70～-60°C for 10 minutes; add dropwise a solution of NFSI in THF (dissolve NFSI (7.35g, 23mmol, 3eq) in THF 70ml)), dropwise, stirred at -70～-60°C for 10 minutes, naturally warmed up to 20～30°C and stirred for 3 hours; added saturated aqueous ammonium chloride solution (80ml) to quench the reaction, and the reaction solution was washed with ethyl acetate ( 150ml*3) extraction, combined organic phases, washed with saturated brine, concentrated to dryness to obtain the crude product, purified by column chromatography, the eluent was petroleum ether: ethyl acetate = 1:0-1:3, to obtain compound A7-3 , yellow solid, 0.85g, yield 31.2%. LC-MS (ESI): [M+1]+=355.8
1H NMR(400MHz，CDCl3)δ11.49(s，1H)，8.64(d，J＝9.2Hz，1H)，7.74(d，J＝8.5Hz， 1H)，6.06(s，1H)，3.01(t，J＝6.4Hz，2H)，2.55-2.39(m，2H)，2.19(s，3H)，1.44(s，9H)1H NMR (400MHz, CDCl3) δ11.49(s, 1H), 8.64(d, J=9.2Hz, 1H), 7.74(d, J=8.5Hz, 1H), 6.06(s, 1H), 3.01(t , J=6.4Hz, 2H), 2.55-2.39(m, 2H), 2.19(s, 3H), 1.44(s, 9H)
于100ml单口烧瓶中，将A7-3化合物(0.85g，2.4mmol)，与6N盐酸(30ml)混合，加热回流反应4小时。停止加热，待反应液冷却至室温后，用碳酸氢钠调节pH至7～8，用二氯甲烷(30ml*3)萃取，合并有机相，饱和食盐水洗涤，浓缩干得粗品，经柱层析纯化，洗脱剂为石油醚∶乙酸乙酯＝1∶0～1∶3，得A7化合物，棕色固体0.25g，收率49.1％。LC-MS(ESI)：[M+1]+＝213.9In a 100ml one-necked flask, compound A7-3 (0.85g, 2.4mmol) was mixed with 6N hydrochloric acid (30ml), and heated to reflux for 4 hours. Stop heating, and after the reaction solution is cooled to room temperature, adjust the pH to 7-8 with sodium bicarbonate, extract with dichloromethane (30ml*3), combine the organic phases, wash with saturated brine, concentrate and dry to obtain the crude product, and pass through the column layer Analysis and purification, the eluent is petroleum ether: ethyl acetate = 1:0 ~ 1:3, to obtain compound A7, brown solid 0.25g, yield 49.1%. LC-MS (ESI): [M+1]+=213.9
实施例1.1.8化合物A8的合成 The synthesis of embodiment 1.1.8 compound A8
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000087
Figure PCTCN2022139765-appb-000087
按照文献Journal of Medicinal Chemistry，1989，vol.32，#6，p.1217-1230方法合成A8-1化合物。LC-MS：(ESI)：[M+1]+＝146.3。Compound A8-1 was synthesized according to the literature Journal of Medicinal Chemistry, 1989, vol.32, #6, p.1217-1230. LC-MS: (ESI): [M+1]+=146.3.
A8-1化合物(1.8g)、DIEA(4.8g)加入DCM(50ml)中。冷至0℃。加入三氟乙酸酐(4.8g)，升温至室温，反应1小时。加入水(20ml)，分层有机层，旋干。过柱纯化(EA∶PE＝0-30％)得A8-2中间体为白色固体，2.7g。LC-MS：(ESI)：[M+1]+＝242.2。Compound A8-1 (1.8 g), DIEA (4.8 g) were added to DCM (50 ml). Cool to 0°C. Trifluoroacetic anhydride (4.8 g) was added, the temperature was raised to room temperature, and the reaction was carried out for 1 hour. Water (20ml) was added, the organic layer was separated and spin-dried. After column purification (EA:PE=0-30%), intermediate A8-2 was obtained as a white solid, 2.7 g. LC-MS: (ESI): [M+1]+=242.2.
Zn(Et) 2(33.6ml)加入DCM(70ml)中，冷至0℃。加入TFA(3.8g)、CH 2I 2(9.0g).反应15分钟。滴加A8-2化合物(2.7g)的DCM(30ml)溶液。滴毕，室温搅拌过夜。加入水(30ml)，分层有机层，干燥，旋干得A8-3中间体为灰白色固体，2.7g。LC-MS(ESI)：[M+1]+＝256.1。 Zn(Et) 2 (33.6ml) was added to DCM (70ml) and cooled to 0°C. Add TFA (3.8 g), CH 2 I 2 (9.0 g). React for 15 minutes. A solution of compound A8-2 (2.7 g) in DCM (30 ml) was added dropwise. After dropping, stir overnight at room temperature. Water (30ml) was added, the organic layer was separated, dried, and spin-dried to give intermediate A8-3 as off-white solid, 2.7g. LC-MS (ESI): [M+1]+=256.1.
A8-3中间体(1.5g)加入DCM(100ml)及乙酸酐(30ml)中，冷至0℃，加入65％硝酸 (2.8g)，室温反应过夜。加入水(50ml)、DCM(100ml)。分层有机层，旋干。油泵拉干得A8-4中间体粗品，1.6g，直接用于下一步。LC-MS(ESI)：[M+1]+＝301.2。A8-3 intermediate (1.5g) was added to DCM (100ml) and acetic anhydride (30ml), cooled to 0°C, 65% nitric acid (2.8g) was added, and reacted overnight at room temperature. Water (50ml), DCM (100ml) were added. Separate the organic layers and spin dry. The crude intermediate of A8-4, 1.6 g, was dried by oil pump, which was directly used in the next step. LC-MS (ESI): [M+1]+=301.2.
A8-4中间体(1.6g粗品)、铁粉(3.2g)、氯化铵(6.4g)加入无水乙醇(100ml)中，升温回流过夜。冷却，过滤，旋干，过柱纯化(EA∶PE＝0-50％)得A8-5中间体为棕色油状物，0.25g。LC-MS(ESI)：[M+1]+＝271.3。A8-4 intermediate (1.6g crude product), iron powder (3.2g), and ammonium chloride (6.4g) were added to absolute ethanol (100ml), and the temperature was raised to reflux overnight. Cooled, filtered, spin-dried, and purified by column (EA:PE=0-50%) to obtain intermediate A8-5 as a brown oil, 0.25g. LC-MS (ESI): [M+1]+=271.3.
A8-5中间体(250mg)、DIEA(400mg)加入DCM(10ml)中，冷至0℃，加入三氟乙酸酐(320mg)，室温反应1小时。加入水(5ml)洗涤。分出有机层，干燥，过滤，旋干得A8-6中间体为黄色固体，340mg。LC-MS(ESI)：[M+1]+＝367.3。Add intermediate A8-5 (250mg) and DIEA (400mg) to DCM (10ml), cool to 0°C, add trifluoroacetic anhydride (320mg), and react at room temperature for 1 hour. Water (5ml) was added for washing. The organic layer was separated, dried, filtered, and spin-dried to give intermediate A8-6 as a yellow solid, 340 mg. LC-MS (ESI): [M+1]+=367.3.
A8-6中间体(150mg)、CrO 3(400mg)加入乙酸(8ml)及乙酸酐(4ml)中，30-40℃反应3小时。旋干溶剂。加入水(5ml)，用DCM(20ml)萃取。分出有机层，干燥，过滤，旋干得A8-7中间体粗品160mg，直接用于下一步。LC-MS(ESI)：[M+1]+＝381.3。 A8-6 intermediate (150mg), CrO 3 (400mg) were added to acetic acid (8ml) and acetic anhydride (4ml), and reacted at 30-40°C for 3 hours. Spin dry solvent. Water (5ml) was added and extracted with DCM (20ml). The organic layer was separated, dried, filtered, and spin-dried to obtain 160 mg of crude intermediate A8-7, which was directly used in the next step. LC-MS (ESI): [M+1]+=381.3.
A8-7中间体经6N盐酸脱出双三氟乙酰基保护，经柱层析纯化得到化合物A8。LC-MS(ESI)：[M+1]+＝189.3。The intermediate A8-7 was deprotected by 6N hydrochloric acid to remove the bistrifluoroacetyl group, and purified by column chromatography to obtain compound A8. LC-MS (ESI): [M+1]+=189.3.
根据上述类似的方法可以合成如下化合物The following compounds can be synthesized according to the above-mentioned similar method
Figure PCTCN2022139765-appb-000088
Figure PCTCN2022139765-appb-000088
Figure PCTCN2022139765-appb-000089
Figure PCTCN2022139765-appb-000089
Figure PCTCN2022139765-appb-000090
Figure PCTCN2022139765-appb-000090
Figure PCTCN2022139765-appb-000091
Figure PCTCN2022139765-appb-000091
实施例组1.2喜树碱类化合物合成(Friedlander反应)Embodiment group 1.2 camptothecin compounds synthesis (Friedlander reaction)
对照喜树碱类化合物MWC-1的合成：Synthesis of the reference camptothecin compound MWC-1:
Figure PCTCN2022139765-appb-000092
Figure PCTCN2022139765-appb-000092
常温下，将化合物A1(176mg，1eq)和CDE环化合物(315mg，1.2eq)、PPTS(301mg，1.2eq)混于甲苯(50ml)中，回流分水反应3～4小时；将反应液直接浓缩干，柱层析纯化，洗脱剂为石油醚洗脱至甲醇/二氯甲烷＝1∶20，得MWC-1，为黄色固体，260mg，LC-MS(ESI)：[M+1]+＝404。At room temperature, compound A1 (176mg, 1eq) and CDE ring compound (315mg, 1.2eq), PPTS (301mg, 1.2eq) were mixed in toluene (50ml), and refluxed for 3-4 hours; the reaction solution was directly Concentrated to dryness, purified by column chromatography, eluting with petroleum ether until methanol/dichloromethane = 1:20, to obtain MWC-1 as a yellow solid, 260 mg, LC-MS (ESI): [M+1] +=404.
实施例1.2.1喜树碱类化合物1的合成 The synthesis of embodiment 1.2.1 camptothecin compound 1
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000093
Figure PCTCN2022139765-appb-000093
于50ml单口反应瓶中，氮气保护下，将化合物A6(120mg，0.59mmol，1eq)、CDE三环化合物(180mg，0.68mmol，1.2eq)和PPTS(35mg，0.14mmol，0.25eq)混合于15ml中，加热回流反应3～4小时；减压蒸馏，蒸除大部分乙酸，残留物经柱层析粗分离，洗脱剂为二氯甲烷∶甲醇＝15∶1，得到粗品80mg，再经制备液相分离纯化，得喜树碱类化合物1为黄色固体，3mg。In a 50ml single-port reaction flask, under nitrogen protection, compound A6 (120mg, 0.59mmol, 1eq), CDE tricyclic compound (180mg, 0.68mmol, 1.2eq) and PPTS (35mg, 0.14mmol, 0.25eq) were mixed in 15ml , heated to reflux for 3 to 4 hours; distilled under reduced pressure to remove most of the acetic acid, and the residue was roughly separated by column chromatography. Liquid phase separation and purification yielded camptothecin compound 1 as a yellow solid, 3 mg.
LC-MS(ESI)：[M+1]+＝432.4。LC-MS (ESI): [M+1]+=432.4.
实施例1.2.2喜树碱类化合物2(2A和2B)的合成 Example 1.2.2 Synthesis of camptothecin compounds 2 (2A and 2B)
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000094
Figure PCTCN2022139765-appb-000094
按照实施例1.2.1类似的方法，用A9代替A6合成得到，经制备液相分离纯化，得化合物2A(保留时间早)，为黄色固体，LC-MS(ESI)：[M+1]+＝418.3；化合物2B(保留时间晚)，为黄色固体，LC-MS(ESI)：[M+1]+＝418.4。According to the method similar to Example 1.2.1, A9 was used instead of A6 to obtain compound 2A (early retention time) as a yellow solid, LC-MS (ESI): [M+1]+ =418.3; compound 2B (late retention time), a yellow solid, LC-MS (ESI): [M+1]+=418.4.
实施例1.2.3喜树碱类化合物3的合成 The synthesis of embodiment 1.2.3 camptothecin compound 3
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000095
Figure PCTCN2022139765-appb-000095
按照实施例1.2.1类似方法合成，用A7代替A6，用甲苯代替乙酸作溶剂，制备得喜 树碱类化合物3为土黄色固体，LC-MS(ESI)：[M+1]+＝440.8Synthesize according to the similar method of Example 1.2.1, replace A6 with A7, replace acetic acid with toluene as solvent, and prepare camptothecin compound 3 as a khaki solid, LC-MS (ESI): [M+1]+=440.8
1H NMR(400MHz，Acetone)δ7.89(d，J＝9.1Hz，1H)，7.48(d，J＝9.1Hz，1H)，7.37(s，1H)，7.31(d，J＝8.9Hz，0H)，5.59-5.26(m，5H)，3.11-3.05(m，4H)，2.73-2.59(m，3H)，1.98(dt，J＝14.0，7.0Hz，1H)，1.05-0.98(m，3H)1H NMR (400MHz, Acetone) δ7.89(d, J=9.1Hz, 1H), 7.48(d, J=9.1Hz, 1H), 7.37(s, 1H), 7.31(d, J=8.9Hz, 0H ), 5.59-5.26(m, 5H), 3.11-3.05(m, 4H), 2.73-2.59(m, 3H), 1.98(dt, J=14.0, 7.0Hz, 1H), 1.05-0.98(m, 3H )
1H NMR(400MHz，DMSO)δ7.87(d，J＝9.1Hz，0H)，7.41(d，J＝9.1Hz，0H)，7.21(s，0H)，6.50(s，0H)，6.15(s，1H)，5.36(d，J＝46.6Hz，1H)，2.93(t，J＝6.5Hz，1H)，2.66-2.54(m，1H)，1.97-1.68(m，3H)，1.24(s，1H)，0.88(t，J＝7.3Hz，3H)1H NMR (400MHz, DMSO) δ7.87(d, J=9.1Hz, 0H), 7.41(d, J=9.1Hz, 0H), 7.21(s, 0H), 6.50(s, 0H), 6.15(s , 1H), 5.36(d, J=46.6Hz, 1H), 2.93(t, J=6.5Hz, 1H), 2.66-2.54(m, 1H), 1.97-1.68(m, 3H), 1.24(s, 1H), 0.88(t, J=7.3Hz, 3H)
实施例1.2.4喜树碱类化合物4(4A和4B)的合成 The synthesis of embodiment 1.2.4 camptothecin compounds 4 (4A and 4B)
Figure PCTCN2022139765-appb-000096
Figure PCTCN2022139765-appb-000096
按照实施例1.2.1类似的合成方法，用A10代替A6制备得到，经制备液相分离纯化，得化合物4A(保留时间早)，为土黄色固体，LC-MS(ESI)：[M+1]+＝422.3；化合物4B(保留时间晚)，为土黄色固体，LC-MS(ESI)：[M+1]+＝422.3。According to the similar synthesis method of Example 1.2.1, A10 was used instead of A6 to obtain compound 4A (early retention time) as a khaki solid, LC-MS (ESI): [M+1 ]+=422.3; compound 4B (late retention time), a khaki solid, LC-MS (ESI): [M+1]+=422.3.
实施例1.2.5喜树碱类化合物5的合成 Embodiment 1.2.5 Synthesis of camptothecin compound 5
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000097
Figure PCTCN2022139765-appb-000097
按照实施例1.2.1类似方法，用A12代替A6制备得到喜树碱类化合物5为黄色固体，LC-MS(ESI)：[M+1]+＝444.3。According to the similar method of Example 1.2.1, A12 was used instead of A6 to prepare camptothecin compound 5 as a yellow solid, LC-MS (ESI): [M+1]+=444.3.
实施例1.2.6喜树碱类化合物6的合成 The synthesis of embodiment 1.2.6 camptothecin compound 6
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000098
Figure PCTCN2022139765-appb-000098
按照实施例1.2.1类似方法，用A11代替A6合成得到，喜树碱类化合物6为黄色固体，LC-MS(ESI)：[M+1]+＝430.5。According to the similar method of Example 1.2.1, it was synthesized by substituting A11 for A6. Camptothecin compound 6 was obtained as a yellow solid, LC-MS (ESI): [M+1]+=430.5.
实施例1.2.7喜树碱类化合物7的合成 Synthesis of Example 1.2.7 Camptothecin Compound 7
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000099
Figure PCTCN2022139765-appb-000099
按照实施例1.2.1类似的合成方法，用A8代替A6合成，得化合物7为黄色固体，0.7mg。LC-MS(ESI)：[M+1]+＝416.3。According to the similar synthesis method of Example 1.2.1, A8 was used instead of A6 to obtain compound 7 as a yellow solid, 0.7 mg. LC-MS (ESI): [M+1]+=416.3.
实施例1.2.8喜树碱类化合物9的合成 The synthesis of embodiment 1.2.8 camptothecin compound 9
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000100
Figure PCTCN2022139765-appb-000100
按照实施例1.2.1类似的合成方法，用A3代替A6合成，得到喜树碱类化合物9，为棕红色固体，LC-MS(ESI)：[M+1]+＝420.3。According to the similar synthesis method of Example 1.2.1, A3 was used instead of A6 to obtain camptothecin compound 9 as a brown-red solid, LC-MS (ESI): [M+1]+=420.3.
实施例1.2.9喜树碱类化合物12的合成 Synthesis of Example 1.2.9 Camptothecin Compound 12
按照与文献Journal of Medicinal chemistry，1998，41(13)，2308-2318类似的方法合成化合物12，合成路线如下：Compound 12 was synthesized according to a method similar to the literature Journal of Medicinal chemistry, 1998, 41 (13), 2308-2318, and the synthetic route is as follows:
Figure PCTCN2022139765-appb-000101
Figure PCTCN2022139765-appb-000101
按照实施例1.2.1类似的合成方法，用A4代替A6，用甲苯代替乙酸作溶剂，合成得化合物12为棕色固体，30mg；LC-MS(ESI)：[M+1]+＝422.5。According to the similar synthesis method of Example 1.2.1, A4 was used instead of A6, and toluene was used instead of acetic acid as solvent to synthesize compound 12 as a brown solid, 30 mg; LC-MS (ESI): [M+1]+=422.5.
实施例1.2.10喜树碱类化合物13的合成 Example 1.2.10 Synthesis of camptothecin compound 13
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000102
Figure PCTCN2022139765-appb-000102
按照实施例1.2.1类似方法的合成方法，用A15代替A6，用甲苯作溶剂合成得到化合物13，为棕色固体，LC-MS(ESI)：[M+1]+＝458.4。According to the synthesis method of Example 1.2.1, A6 was replaced by A15, and toluene was used as a solvent to synthesize compound 13 as a brown solid, LC-MS (ESI): [M+1]+=458.4.
实施例1.2.11喜树碱类化合物14的合成 The synthesis of embodiment 1.2.11 camptothecin compound 14
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000103
Figure PCTCN2022139765-appb-000103
按照实施例1.2.1类似方法的合成方法，用A5代替A6，用甲苯作溶剂，制备得到化合物14，为黄色固体，LC-MS(ESI)：[M+1]+＝406.1。According to the synthesis method similar to Example 1.2.1, A5 was used instead of A6, and toluene was used as solvent to prepare compound 14 as a yellow solid, LC-MS (ESI): [M+1]+=406.1.
实施例1.2.12喜树碱类化合物14-P的合成 The synthesis of embodiment 1.2.12 camptothecin compound 14-P
Figure PCTCN2022139765-appb-000104
Figure PCTCN2022139765-appb-000104
常温下，将化合物12(200mg)溶于乙酸(24ml)、水(3ml)混合溶剂中，氮气保护下，降温到5℃，加入30％H 2O 2(580mg)，搅拌反应2小时，反应液浓缩干，用HPLC制备液相分离得到化合物14-P1和14-P2。冻干得到化合物14-P1棕色固体42mg、化合物14-P2棕色固体25mg；LC-MS(ESI)：[M+1]+＝438.5。 At room temperature, compound 12 (200 mg) was dissolved in a mixed solvent of acetic acid (24 ml) and water (3 ml), under the protection of nitrogen, the temperature was lowered to 5 ° C, 30% H 2 O 2 (580 mg) was added, and the reaction was stirred for 2 hours. The solution was concentrated to dryness and separated by HPLC to obtain compounds 14-P1 and 14-P2. Freeze-drying gave 42 mg of compound 14-P1 as a brown solid and 25 mg of compound 14-P2 as a brown solid; LC-MS (ESI): [M+1]+=438.5.
实施例1.2.13喜树碱类化合物15的合成 Synthesis of Example 1.2.13 Camptothecin Compound 15
Figure PCTCN2022139765-appb-000105
Figure PCTCN2022139765-appb-000105
常温下，将化合物12(100mg)溶于乙酸(24ml)、水(3ml)混合溶剂中，氮气保护下，降温到5℃，加入30％H 2O 2(750mg)，搅拌反应2小时，回到室温25℃搅拌反应过夜，LCMS分析反应完全，将反应液浓缩干，用HPLC制备液相分离，收集，冻干得到化合物15为棕色固体8mg。LC-MS(ESI)：[M+1]+＝454.4。 Dissolve compound 12 (100mg) in a mixed solvent of acetic acid (24ml) and water (3ml) at room temperature, under nitrogen protection, cool down to 5°C, add 30% H 2 O 2 (750mg), stir for 2 hours, return to The reaction was stirred at room temperature 25°C overnight, and the reaction was complete by LCMS analysis. The reaction solution was concentrated to dryness, separated by HPLC, collected, and lyophilized to obtain 8 mg of compound 15 as a brown solid. LC-MS (ESI): [M+1]+=454.4.
实施例1.2.14喜树碱类化合物16的合成 The synthesis of embodiment 1.2.14 camptothecin compound 16
Figure PCTCN2022139765-appb-000106
Figure PCTCN2022139765-appb-000106
按照WO2020200880A1专利方法合成中间体16a，然后按照文献(J.Med.Chem.2008，51，3040-3044)的方法，制备16a-硫代喜树碱结构中间体16c，最后脱除乙酰基保护，经制备得到化合物16，LC-MS(ESI)：[M+1]+＝420.3。Synthesize intermediate 16a according to WO2020200880A1 patent method, and then prepare 16a-thiocamptothecin structure intermediate 16c according to the method of literature (J.Med.Chem.2008, 51, 3040-3044), and finally remove the acetyl group protection, Compound 16 was prepared, LC-MS (ESI): [M+1]+=420.3.
实施例1.2.15喜树碱类化合物17的合成 Example 1.2.15 Synthesis of Camptothecin Compound 17
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000107
Figure PCTCN2022139765-appb-000107
按照实施例1.2.1类似方法的合成方法，用A1代替A6，用HCDE代替CDE，用甲苯作溶剂合成，得到高喜树碱化合物17(S，R混合物)，LC-MS(ESI)：[M+1]+＝418.5。According to the synthetic method of the similar method in Example 1.2.1, replace A6 with A1, replace CDE with HCDE, and synthesize with toluene as solvent to obtain homocamptothecin compound 17 (S, R mixture), LC-MS (ESI): [M+ 1]+=418.5.
实施例1.2.16喜树碱类化合物18的合成 Embodiment 1.2.16 Synthesis of camptothecin compound 18
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000108
Figure PCTCN2022139765-appb-000108
按照实施例1.2.1类似方法的合成方法，用A16代替A6，用甲苯作溶剂合成化合物18，为淡黄色固体，LC-MS(ESI)：[M+1]+＝424.2。According to the synthesis method of Example 1.2.1, A6 was replaced by A16, and compound 18 was synthesized using toluene as a solvent. It was a light yellow solid, LC-MS (ESI): [M+1]+=424.2.
实施例1.2.17喜树碱类化合物19的合成 Example 1.2.17 Synthesis of Camptothecin Compound 19
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000109
Figure PCTCN2022139765-appb-000109
按照实施例1.2.1类似方法的合成方法，用A17代替A6，用甲苯作溶剂合成化合物19，为黄色固体，LC-MS(ESI)：[M+1]+＝460.5。According to the synthesis method of Example 1.2.1, A6 was replaced by A17, and compound 19 was synthesized by using toluene as a solvent. It was a yellow solid, LC-MS (ESI): [M+1]+=460.5.
实施例1.2.18喜树碱类化合物20的合成 The synthesis of embodiment 1.2.18 camptothecin compound 20
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000110
Figure PCTCN2022139765-appb-000110
按照实施例1.2.1类似方法的合成方法，用A13代替A6，用甲苯作溶剂合成化合物20，为黄色固体，LC-MS(ESI)：[M+1]+＝458.2。According to the synthesis method of Example 1.2.1, A6 was replaced by A13, and compound 20 was synthesized by using toluene as a solvent. It was a yellow solid, LC-MS (ESI): [M+1]+=458.2.
实施例1.2.19喜树碱类化合物21的合成 Example 1.2.19 Synthesis of Camptothecin Compound 21
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000111
Figure PCTCN2022139765-appb-000111
按照实施例1.2.1类似方法的合成方法，用A24代替A6，用甲苯作溶剂，得到化合物21，为棕红色固体，LC-MS(ESI)：[M+1]+＝418.4。According to the synthesis method of Example 1.2.1, A6 was replaced by A24, and toluene was used as solvent to obtain compound 21 as a brownish-red solid, LC-MS (ESI): [M+1]+=418.4.
实施例1.2.20喜树碱类化合物22的合成 The synthesis of embodiment 1.2.20 camptothecin compound 22
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000112
Figure PCTCN2022139765-appb-000112
按照实施例1.2.1类似方法的合成方法，用A25代替A6，用甲苯作溶剂，得到化合物22，为棕红色固体，LC-MS(ESI)：[M+1]+＝434.4。According to the synthesis method of Example 1.2.1, A6 was replaced by A25, and toluene was used as solvent to obtain compound 22 as a brownish-red solid, LC-MS (ESI): [M+1]+=434.4.
实施例1.2.21喜树碱类化合物23的合成 Example 1.2.21 Synthesis of camptothecin compound 23
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000113
Figure PCTCN2022139765-appb-000113
按照实施例1.2.1类似方法的合成方法，用A21代替A6，用甲苯作溶剂，得到化合物23，为棕红色固体，LC-MS(ESI)：[M+1]+＝432.4。According to the synthesis method of Example 1.2.1, A6 was replaced by A21, and toluene was used as solvent to obtain compound 23 as a brownish-red solid, LC-MS (ESI): [M+1]+=432.4.
实施例1.2.22喜树碱类化合物24的合成 Example 1.2.22 Synthesis of camptothecin compound 24
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000114
Figure PCTCN2022139765-appb-000114
按照实施例1.2.1类似方法的合成方法，用A20代替A6，用甲苯作溶剂，得到化合物24(一对异构体)，为棕红色固体，LC-MS(ESI)：[M+1]+＝418.4。According to the synthesis method of the similar method in Example 1.2.1, replace A6 with A20, and use toluene as solvent to obtain compound 24 (a pair of isomers) as a brownish red solid, LC-MS (ESI): [M+1] +=418.4.
实施例1.2.23喜树碱类化合物25的合成 Synthesis of Example 1.2.23 Camptothecin Compound 25
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000115
Figure PCTCN2022139765-appb-000115
按照实施例1.2.1类似方法的合成方法，用A32代替A6，用甲苯作溶剂，得到化合物25，为棕红色固体，LC-MS(ESI)：[M+1]+＝432.4。According to the synthesis method of Example 1.2.1, A6 was replaced by A32, and toluene was used as solvent to obtain compound 25 as a brownish-red solid, LC-MS (ESI): [M+1]+=432.4.
实施例1.2.24喜树碱类化合物26的合成 The synthesis of embodiment 1.2.24 camptothecin compound 26
Figure PCTCN2022139765-appb-000116
Figure PCTCN2022139765-appb-000116
按照实施例1.2.1类似方法的合成方法，用A18代替A6，用甲苯作溶剂合成，得到化合物26，为棕红色固体，LC-MS(ESI)：[M+1]+＝440.4。According to the synthesis method similar to Example 1.2.1, A6 was replaced by A18 and synthesized with toluene as a solvent to obtain compound 26 as a brownish-red solid, LC-MS (ESI): [M+1]+=440.4.
实施例1.2.25喜树碱类化合物27的合成 Example 1.2.25 Synthesis of camptothecin compound 27
Figure PCTCN2022139765-appb-000117
Figure PCTCN2022139765-appb-000117
按照实施例1.2.1类似方法的合成方法，用A19代替A6，用甲苯作溶剂，得到化合物27，为棕红色固体，LC-MS(ESI)：[M+1]+＝476.4。According to the synthesis method of Example 1.2.1, A6 was replaced by A19, and toluene was used as solvent to obtain compound 27 as a brownish-red solid, LC-MS (ESI): [M+1]+=476.4.
实施例1.2.26喜树碱类化合物31的合成 Example 1.2.26 Synthesis of camptothecin compound 31
Figure PCTCN2022139765-appb-000118
Figure PCTCN2022139765-appb-000118
按照实施例1.2.1类似方法的合成方法，用A14代替A6，用甲苯作溶剂得到化合物31，为黄色固体，LC-MS(ESI)：[M+1]+＝442.4。According to the synthesis method of Example 1.2.1, A6 was replaced by A14, and toluene was used as solvent to obtain compound 31 as a yellow solid, LC-MS (ESI): [M+1]+=442.4.
实施例1.2.27喜树碱类化合物32的合成 Example 1.2.27 Synthesis of Camptothecin Compound 32
Figure PCTCN2022139765-appb-000119
Figure PCTCN2022139765-appb-000119
按照实施例1.2.1类似方法的合成方法，用A2代替A6，用HCDE代替CDE，用甲苯作溶剂合成得到化合物32，为黄色固体，LC-MS(ESI)：[M+1]+＝436.4。According to the synthesis method of the similar method in Example 1.2.1, A2 was used to replace A6, HCDE was used to replace CDE, and toluene was used as a solvent to synthesize compound 32 as a yellow solid, LC-MS (ESI): [M+1]+=436.4 .
实施例1.2.28喜树碱类化合物33的合成 Example 1.2.28 Synthesis of camptothecin compound 33
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000120
Figure PCTCN2022139765-appb-000120
按照实施例1.2.1类似方法的合成方法，用A23代替A6，用甲苯作溶剂，化合物33为黄色固体，LC-MS(ESI)：[M+1]+＝458.5。According to the synthesis method of Example 1.2.1, replacing A6 with A23 and using toluene as solvent, compound 33 was a yellow solid, LC-MS (ESI): [M+1]+=458.5.
实施例1.2.29喜树碱类化合物34的合成 Example 1.2.29 Synthesis of camptothecin compound 34
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000121
Figure PCTCN2022139765-appb-000121
按照实施例1.2.1类似方法的合成方法，用A22代替A6，用甲苯作溶剂，合成化合物34为黄色固体，LC-MS(ESI)：[M+1]+＝458.5。According to the synthesis method of Example 1.2.1, A6 was replaced by A22, and toluene was used as solvent to synthesize compound 34 as a yellow solid, LC-MS (ESI): [M+1]+=458.5.
实施例1.2.30喜树碱类化合物35和化合物38的合成 Example 1.2.30 Synthesis of camptothecin compound 35 and compound 38
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000122
Figure PCTCN2022139765-appb-000122
按照实施例1.2.1类似方法的合成方法，用A26代替A6，用甲苯作溶剂，合成化合 物35，为棕红色固体，LC-MS(ESI)：[M+1]+＝438.7。According to the synthesis method of Example 1.2.1, A6 was replaced by A26, and toluene was used as solvent to synthesize compound 35 as a brownish-red solid, LC-MS (ESI): [M+1]+=438.7.
按照实施例1.2.1类似方法的合成方法，用A27代替A6，用甲苯作溶剂，合成，得到化合物38，为棕红色固体，LC-MS(ESI)：[M+1]+＝474.7。According to the synthesis method similar to Example 1.2.1, A6 was replaced by A27, and toluene was used as solvent to synthesize to obtain compound 38 as a brownish-red solid, LC-MS (ESI): [M+1]+=474.7.
实施例1.2.31喜树碱类化合物36和化合物39的合成 Example 1.2.31 Synthesis of camptothecin compound 36 and compound 39
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000123
Figure PCTCN2022139765-appb-000123
按照实施例1.2.1类似方法的合成方法，用A30代替A6，用甲苯作溶剂，合成化合物36，为棕红色固体，LC-MS(ESI)：[M+1]+＝456.7。According to the synthesis method of Example 1.2.1, A6 was replaced by A30, and toluene was used as solvent to synthesize compound 36 as a brownish-red solid, LC-MS (ESI): [M+1]+=456.7.
按照实施例1.2.1类似方法的合成方法，用A31代替A6，用甲苯作溶剂，合成类得到化合物39，为棕红色固体，LC-MS(ESI)：[M+1]+＝492.7According to the synthesis method of the similar method in Example 1.2.1, replace A6 with A31, and use toluene as solvent to synthesize compound 39 as a brownish red solid, LC-MS (ESI): [M+1]+=492.7
实施例1.2.32喜树碱类化合物37和化合物40的合成 Example 1.2.32 Synthesis of camptothecin compound 37 and compound 40
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000124
Figure PCTCN2022139765-appb-000124
按照实施例1.2.1类似方法的合成方法，用A28代替A6，用甲苯作溶剂，合成化合物37，为棕红色固体，LC-MS(ESI)：[M+1]+＝440.8。According to the synthesis method of Example 1.2.1, replacing A6 with A28 and using toluene as solvent, compound 37 was synthesized as a brownish-red solid, LC-MS (ESI): [M+1]+=440.8.
按照实施例1.2.1类似方法的合成方法，用A29代替A6，用甲苯作溶剂，合成得到化合物40，为棕红色固体，LC-MS(ESI)：[M+1]+＝476.7。According to the synthesis method similar to Example 1.2.1, A29 was used instead of A6, and toluene was used as solvent to synthesize compound 40 as a brownish-red solid, LC-MS (ESI): [M+1]+=476.7.
实施例组2含药连接子的合成The synthesis of embodiment group 2 drug-containing linkers
实施例2.1含药连接子MWD-L1的合成 Example 2.1 Synthesis of drug-containing linker MWD-L1
Figure PCTCN2022139765-appb-000125
Figure PCTCN2022139765-appb-000125
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000126
Figure PCTCN2022139765-appb-000126
GGFG-Dxd按照专利公开文件(US20190151328A1)中所述方法合成：GGFG-Dxd is synthesized according to the method described in the patent publication (US20190151328A1):
Figure PCTCN2022139765-appb-000127
Figure PCTCN2022139765-appb-000127
GGFG-Dxd为淡黄色固体，LC-MS(ESI)：M+1＝841；GGFG-Dxd is light yellow solid, LC-MS (ESI): M+1=841;
BL接头化合物按照专利公布文件WO2018/095422A1中所述方法合成：The BL linker compound was synthesized according to the method described in the patent publication WO2018/095422A1:
Figure PCTCN2022139765-appb-000128
Figure PCTCN2022139765-appb-000128
BL接头化合物为黄色固体，LC-MS(ESI)：M+1＝858The BL linker compound is a yellow solid, LC-MS (ESI): M+1=858
BL接头化合物(857mg，1mmol)，GGFG-Dxd(840mg，1mmol，1eq)，DIPEA(323mg，2.5mmol，2.5eq)，HATU(570mg，1.5mmol，1.5eq)，溶于30ml DCM中，搅拌反应2h。将反应液冷却至5～10℃，加入1N盐酸(20ml)，搅拌0.5h。分液，水相用DCM(30ml*2)萃取，合并有机相。有机相用饱和食盐水洗涤，无水硫酸钠干燥，抽滤，旋干。柱层析纯化，洗脱剂：DCM∶MeOH＝50∶1～10∶1，得含药接头MWD-L1为黄色固体，500mg，收率29.8％，LC-MS(ESI)：M+1＝1681，(M+1)/2＝840.9。BL linker compound (857mg, 1mmol), GGFG-Dxd (840mg, 1mmol, 1eq), DIPEA (323mg, 2.5mmol, 2.5eq), HATU (570mg, 1.5mmol, 1.5eq), dissolved in 30ml DCM, stirred reaction 2h. The reaction solution was cooled to 5-10°C, 1N hydrochloric acid (20ml) was added, and stirred for 0.5h. The layers were separated, the aqueous phase was extracted with DCM (30ml*2), and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered with suction, and spin-dried. Purified by column chromatography, eluent: DCM:MeOH=50:1～10:1, the drug-containing linker MWD-L1 was obtained as a yellow solid, 500 mg, yield 29.8%, LC-MS (ESI): M+1= 1681, (M+1)/2=840.9.
实施例2.2含药连接子MWC-L2的合成 Example 2.2 Synthesis of drug-containing linker MWC-L2
Figure PCTCN2022139765-appb-000129
Figure PCTCN2022139765-appb-000129
步骤1step 1
Boc-Val-Ala-OH(288mg，1mmol)，A1化合物(176mg，1mmol，1eq)，DIPEA(322mg，2.5mmol，2.5eq)，HATU(456mg，1.2mmol，1.2eq)溶于30ml DCM中，搅拌反应2h。反应液旋干，柱层析纯化，洗脱剂：PE∶EA＝10∶1～5∶1，定量地得到中间体2-1为灰绿色固体450mg。Boc-Val-Ala-OH (288mg, 1mmol), A1 compound (176mg, 1mmol, 1eq), DIPEA (322mg, 2.5mmol, 2.5eq), HATU (456mg, 1.2mmol, 1.2eq) were dissolved in 30ml DCM, The reaction was stirred for 2h. The reaction solution was spin-dried and purified by column chromatography, eluent: PE:EA=10:1-5:1, and intermediate 2-1 was quantitatively obtained as 450 mg of gray-green solid.
步骤2 step 2
中间体2-1(450mg，1mmol)，CDE三环化合物(263mg，1mmol，1eq)，对甲苯磺酸吡啶盐(PPTS)(251mg，1mmol，1eq)悬浮于30ml甲苯中，加热回流反应2小时。停止加热，待反应液冷却后，收集固体沉淀物即为中间体2-2粗品，棕色固体750mg。LC-MS(ESI)：M+1＝574。不经纯化直接下一步反应。Intermediate 2-1 (450mg, 1mmol), CDE tricyclic compound (263mg, 1mmol, 1eq), pyridinium p-toluenesulfonate (PPTS) (251mg, 1mmol, 1eq) were suspended in 30ml of toluene, heated to reflux for 2 hours . Heating was stopped, and after the reaction solution was cooled, the solid precipitate was collected, which was the crude product of Intermediate 2-2, 750 mg of brown solid. LC-MS (ESI): M+1=574. The next reaction was carried out directly without purification.
步骤3 step 3
BL接头化合物(857mg，1mmol)，HoSu(138mg，1.2mmol，1.2eq)，DCC(310mg，1.5mmol，1.5eq)溶于30ml DCM中，室温搅拌3h，反应液抽滤，滤液即为A-Osu的DCM溶液。将滤液加入到中间体2-2粗品、DIPEA(323mg，2.5mmol，2.5eq)、DCM(30ml)的混合溶液中，搅拌反应3h。将反应液冷却至10℃下，加入1N盐酸(20ml)，搅拌0.5h。分液，水相用DCM(30ml*2)萃取，合并有机相。有机相用饱和食盐水洗涤，无水硫酸 钠干燥，抽滤，旋干。柱层析纯化，洗脱剂：DCM∶MeOH＝50∶1～10∶1，得连接子MWC-L2为橘红色固体，325mg，收率23.0％，LC-MS(ESI)：M+1＝1414，(M+1)/2＝707BL linker compound (857mg, 1mmol), HoSu (138mg, 1.2mmol, 1.2eq), DCC (310mg, 1.5mmol, 1.5eq) were dissolved in 30ml DCM, stirred at room temperature for 3h, the reaction solution was suction filtered, and the filtrate was A- DCM solution of Osu. The filtrate was added to the mixed solution of the crude intermediate 2-2, DIPEA (323 mg, 2.5 mmol, 2.5 eq), and DCM (30 ml), and the reaction was stirred for 3 h. The reaction solution was cooled to 10°C, 1N hydrochloric acid (20ml) was added, and stirred for 0.5h. The layers were separated, the aqueous phase was extracted with DCM (30ml*2), and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered with suction, and spin-dried. Purified by column chromatography, eluent: DCM:MeOH=50:1～10:1, the linker MWC-L2 was obtained as orange-red solid, 325 mg, yield 23.0%, LC-MS (ESI): M+1= 1414, (M+1)/2=707
实施例2.3含药连接子MWC-L3的合成 Example 2.3 Synthesis of drug-containing linker MWC-L3
Figure PCTCN2022139765-appb-000130
Figure PCTCN2022139765-appb-000130
步骤1step 1
Boc-Gly-OH(175mg，1mmol)，A1化合物(176mg，1mmol，1eq)，DIPEA(322mg，2.5mmol，2.5eq)，HATU(456mg，1.2mmol，1.2eq)溶于30ml DCM中，搅拌反应2h。反应液旋干，柱层析纯化，洗脱剂：PE∶EA＝10∶1～5∶1，定量地得到中间体3-1为淡绿色固体335mg。Boc-Gly-OH (175mg, 1mmol), A1 compound (176mg, 1mmol, 1eq), DIPEA (322mg, 2.5mmol, 2.5eq), HATU (456mg, 1.2mmol, 1.2eq) were dissolved in 30ml DCM, and the reaction was stirred 2h. The reaction solution was spin-dried and purified by column chromatography, eluent: PE:EA=10:1-5:1, and intermediate 3-1 was quantitatively obtained as 335 mg of light green solid.
步骤2 step 2
中间体3-1(335mg，1mmol)，CDE三环化合物(263mg，1mmol，1eq)，PPTS(251mg，1mmol，1eq)悬浮于30ml甲苯中，加热回流反应2小时。停止加热，待反应液冷却后，收集固体沉淀物即为中间体3-2粗品，棕色固体560mg。LC-MS(ESI)：M+1＝461。不经纯化直接下一步反应。Intermediate 3-1 (335mg, 1mmol), CDE tricyclic compound (263mg, 1mmol, 1eq), PPTS (251mg, 1mmol, 1eq) were suspended in 30ml of toluene, heated to reflux for 2 hours. Heating was stopped, and after the reaction liquid was cooled, the solid precipitate was collected, which was the crude product of Intermediate 3-2, 560 mg of brown solid. LC-MS (ESI): M+1=461. The next reaction was carried out directly without purification.
步骤3 step 3
Fmoc-Gly-Gly-Phe-OH(501mg，1mmol)，中间体3-2粗品(560mg)，DIPEA(322mg，2.5mmol，2.5eq)，HATU(456mg，1.2mmol，1.2eq)，溶于20ml DMF中，搅拌反应3h。加入1N盐酸(30ml)，搅拌0.5h。用DCM(30ml*2)萃取，合并有机相。有机相用饱和食盐水洗涤，无水硫酸钠干燥，抽滤，旋干。柱层析纯化，洗脱剂：DCM∶MeOH＝50∶1～10∶1，得中间体3-3为棕色固体，350mg，LC-MS(ESI)：M+1＝945Fmoc-Gly-Gly-Phe-OH (501mg, 1mmol), intermediate 3-2 crude (560mg), DIPEA (322mg, 2.5mmol, 2.5eq), HATU (456mg, 1.2mmol, 1.2eq), dissolved in 20ml In DMF, the reaction was stirred for 3h. Add 1N hydrochloric acid (30ml) and stir for 0.5h. Extract with DCM (30ml*2) and combine the organic phases. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered with suction, and spin-dried. Purified by column chromatography, eluent: DCM:MeOH=50:1～10:1, intermediate 3-3 was obtained as a brown solid, 350 mg, LC-MS (ESI): M+1=945
步骤4 step 4
中间体3-3(350mg，0.37mmol)溶于甲醇(20ml)中，加入二乙胺(2ml)，搅拌反应3h。旋干反应液，得中间体3-4粗品为棕色粘稠物，LC-M(ESI)：M+1＝722，粗品不经纯化直接下一步。Intermediate 3-3 (350mg, 0.37mmol) was dissolved in methanol (20ml), diethylamine (2ml) was added, and the reaction was stirred for 3h. The reaction solution was spin-dried to obtain the crude intermediate 3-4 as a brown viscous product, LC-M (ESI): M+1=722, and the crude product was directly used in the next step without purification.
步骤5 step 5
BL接头化合物(318mg，0.37mmol)，HoSu(51mg，0.44mmol，1.2eq)，DCC(114mg，0.56mmol，1.5eq)溶于30ml DCM中，室温搅拌3h，反应液抽滤。将滤液加入到中间体3-4粗品、DIPEA(120mg，0.93mmol，2.5eq)、DCM(30ml)的混合溶液中，搅拌反应3h。将反应液冷却至10℃下，加入1N盐酸(20ml)，搅拌0.5h。分液，水相用DCM(30ml*2)萃取，合并有机相。有机相用饱和食盐水洗涤，无水硫酸钠干燥，抽滤，旋干。柱层析纯化，洗脱剂：DCM∶MeOH＝50∶1～10∶1，得连接子MWC-L3为橘红色固体120mg，收率20.8％，LC-MS(ESI)：M+1＝1562，(M+1)/2＝781BL linker compound (318mg, 0.37mmol), HoSu (51mg, 0.44mmol, 1.2eq), DCC (114mg, 0.56mmol, 1.5eq) were dissolved in 30ml DCM, stirred at room temperature for 3h, and the reaction solution was suction filtered. The filtrate was added to the mixed solution of the crude intermediate 3-4, DIPEA (120mg, 0.93mmol, 2.5eq), DCM (30ml), and stirred for 3h. The reaction solution was cooled to 10°C, 1N hydrochloric acid (20ml) was added, and stirred for 0.5h. The layers were separated, the aqueous phase was extracted with DCM (30ml*2), and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered with suction, and spin-dried. Purified by column chromatography, eluent: DCM:MeOH=50:1～10:1, the linker MWC-L3 was obtained as orange-red solid 120 mg, yield 20.8%, LC-MS (ESI): M+1=1562 , (M+1)/2=781
实施例2.4含药连接MWE-L4的合成 Example 2.4 Synthesis of drug-linked MWE-L4
Figure PCTCN2022139765-appb-000131
Figure PCTCN2022139765-appb-000131
根据实施例2.2的方法用A3代替A1合成得到MWE-L4，为橘红色固体，LC-MS(ESI)：M+1＝1430According to the method of Example 2.2, A3 was used instead of A1 to synthesize MWE-L4 as an orange-red solid, LC-MS (ESI): M+1=1430
实施例2.5含药连接子MWG-L5的合成 Example 2.5 Synthesis of drug-containing linker MWG-L5
Figure PCTCN2022139765-appb-000132
Figure PCTCN2022139765-appb-000132
根据实施例2.2的方法用A6代替A1合成得到MWG-L5，为橘红色固体，LC-MS(ESI)：M+1＝1442According to the method of Example 2.2, MWG-L5 was synthesized by replacing A1 with A6, which was an orange-red solid, LC-MS (ESI): M+1=1442
实施例2.6含药连接子MWF-L6的合成 Example 2.6 Synthesis of drug-containing linker MWF-L6
Figure PCTCN2022139765-appb-000133
Figure PCTCN2022139765-appb-000133
根据实施例2.2的方法用A7代替A1合成得到MWF-L6，为橘红色固体，LC-MS(ESI)：M+1＝1450According to the method of Example 2.2, MWF-L6 was synthesized by replacing A1 with A7, which was an orange-red solid, LC-MS (ESI): M+1=1450
实施例2.7含药连接子MWF-L7的合成 Example 2.7 Synthesis of drug-containing linker MWF-L7
Figure PCTCN2022139765-appb-000134
Figure PCTCN2022139765-appb-000134
根据实施例2.2的方法用A7代替A1，用Alloc-Ala-Ala-Ala-OH代替Alloc-Val-Ala-OH合成得到MWF-L7，为橘红色固体，LC-MS(ESI)：M+1＝1493.5According to the method of Example 2.2, A7 was used to replace A1, and Alloc-Ala-Ala-Ala-OH was used to replace Alloc-Val-Ala-OH to synthesize MWF-L7 as an orange solid, LC-MS (ESI): M+1 =1493.5
实施例2.8含药连接子MWF-L8的合成 Example 2.8 Synthesis of drug-containing linker MWF-L8
Figure PCTCN2022139765-appb-000135
Figure PCTCN2022139765-appb-000135
根据实施例2.3的方法用A7代替A1合成得到MWF-L8，为橘红色固体，LC-MS(ESI)：M+1＝1598.4。According to the method of Example 2.3, MWF-L8 was synthesized by substituting A7 for A1 to obtain MWF-L8 as an orange-red solid, LC-MS (ESI): M+1=1598.4.
实施例2.9含药连接子MWF-L9的合成 Example 2.9 Synthesis of drug-containing linker MWF-L9
Figure PCTCN2022139765-appb-000136
Figure PCTCN2022139765-appb-000136
根据实施例2.2的方法用A5代替A1合成得到MWF-L9，为黄色固体，LC-MS(ESI)：M+1＝1416.4。According to the method of Example 2.2, MWF-L9 was synthesized by substituting A5 for A1 to obtain MWF-L9 as a yellow solid, LC-MS (ESI): M+1=1416.4.
实施例2.10含药连接子MWF-L10的合成 Example 2.10 Synthesis of drug-containing linker MWF-L10
Figure PCTCN2022139765-appb-000137
Figure PCTCN2022139765-appb-000137
根据实施例2.2的方法用A13代替A1合成得到MWF-L10，为黄色固体， LC-MS(ESI)：M+1＝1468.4。According to the method of Example 2.2, MWF-L10 was synthesized by substituting A13 for A1 to obtain MWF-L10 as a yellow solid, LC-MS (ESI): M+1=1468.4.
实施例2.11含药连接子MWF-L11的合成 Example 2.11 Synthesis of drug-containing linker MWF-L11
Figure PCTCN2022139765-appb-000138
Figure PCTCN2022139765-appb-000138
根据实施例2.2的方法用A18代替A1合成得到MWF-L11，为黄色固体，LC-MS(ESI)：M+1＝1450.4。According to the method of Example 2.2, MWF-L11 was synthesized by substituting A18 for A1 to obtain MWF-L11 as a yellow solid, LC-MS (ESI): M+1=1450.4.
实施例2.12含药连接子MWF-L12的合成 Example 2.12 Synthesis of drug-containing linker MWF-L12
Figure PCTCN2022139765-appb-000139
Figure PCTCN2022139765-appb-000139
根据实施例2.2的方法用A16代替A1合成得到MWF-L12，为黄色固体，LC-MS(ESI)：M+1＝1434.4。According to the method of Example 2.2, MWF-L12 was synthesized by substituting A16 for A1 to obtain MWF-L12 as a yellow solid, LC-MS (ESI): M+1=1434.4.
实施例2.13含药连接子MWF-L13的合成 Example 2.13 Synthesis of drug-containing linker MWF-L13
Figure PCTCN2022139765-appb-000140
Figure PCTCN2022139765-appb-000140
根据实施例2.2的方法用A19代替A1合成得到MWF-L13，为黄色固体，LC-MS(ESI)：M+1＝1486.4。According to the method of Example 2.2, MWF-L13 was synthesized by substituting A19 for A1 to obtain MWF-L13 as a yellow solid, LC-MS (ESI): M+1=1486.4.
实施例2.14含药连接子MWF-L14的合成 Example 2.14 Synthesis of drug-containing linker MWF-L14
Figure PCTCN2022139765-appb-000141
Figure PCTCN2022139765-appb-000141
根据实施例2.2的方法用A17代替A1合成得到MWF-L14，为黄色固体，LC-MS(ESI)：M+1＝1470.4。According to the method of Example 2.2, MWF-L14 was synthesized by substituting A17 for A1 to obtain MWF-L14 as a yellow solid, LC-MS (ESI): M+1=1470.4.
实施例2.15含药连接子MWF-L15的合成 Example 2.15 Synthesis of drug-containing linker MWF-L15
Figure PCTCN2022139765-appb-000142
Figure PCTCN2022139765-appb-000142
Figure PCTCN2022139765-appb-000143
Figure PCTCN2022139765-appb-000143
根据实施例2.2的方法用A2代替A1合成得到MWF-L15，为黄色固体，LC-MS(ESI)：M+1＝1432.4。According to the method of Example 2.2, MWF-L15 was synthesized by substituting A2 for A1 to obtain MWF-L15 as a yellow solid, LC-MS (ESI): M+1=1432.4.
实施例2.16含药连接子MWD-L7的合成 Example 2.16 Synthesis of drug-containing linker MWD-L7
Figure PCTCN2022139765-appb-000144
Figure PCTCN2022139765-appb-000144
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000145
Figure PCTCN2022139765-appb-000145
步骤1step 1
Fmoc-Val-Cit-PAB-PNP(153mg，0.2mmol)，甲磺酸依沙替康(106mg，0.2mmol，1eq)，DIPEA(65mg，0.5mmol，2.5eq)，溶于30ml DCM中，搅拌反应2h。反应液旋干，柱层析纯化，洗脱剂：DCM∶MeOH＝50∶1～10∶1，得到中间体5-1为淡黄色固体180mg。LC-MS(ESI)：M+1＝1064。Fmoc-Val-Cit-PAB-PNP (153mg, 0.2mmol), exatecan mesylate (106mg, 0.2mmol, 1eq), DIPEA (65mg, 0.5mmol, 2.5eq), dissolved in 30ml DCM, stirred Reaction 2h. The reaction solution was spin-dried and purified by column chromatography, eluent: DCM:MeOH=50:1-10:1, to obtain 180 mg of intermediate 5-1 as a pale yellow solid. LC-MS (ESI): M+1=1064.
步骤2 step 2
中间体5-1(180mg，0.17mmol)溶于甲醇(20ml)中，加入二乙胺(2ml)，搅拌反应3h。旋干反应液，得中间体5-2粗品为淡黄色粘稠物，LC-M(ESI)：M+1＝842，粗品不经纯化直接下一步。Intermediate 5-1 (180mg, 0.17mmol) was dissolved in methanol (20ml), diethylamine (2ml) was added, and the reaction was stirred for 3h. The reaction solution was spin-dried to obtain the crude intermediate 5-2 as light yellow viscous substance, LC-M (ESI): M+1=842, and the crude product was directly used in the next step without purification.
步骤3 step 3
BL接头化合物(146mg，0.17mmol)，HoSu(25mg，0.21mmol，1.2eq)，DCC(54mg，0.26mmol，1.5eq)溶于20ml DCM中，室温搅拌3h，反应液抽滤。将滤液加入到中间体5-1粗品、DIPEA(56mg，0.43mmol，2.5eq)、DCM(20ml)的混合溶液中，搅拌反应3h。将反应液冷却至5～10℃，加入1N盐酸(20ml)，搅拌0.5h。分液，水相用DCM(30ml*2)萃取，合并有机相。有机相用饱和食盐水洗涤，无水硫酸钠干燥，抽滤，旋干。柱层析纯化，洗脱剂：DCM∶MeOH＝50∶1～10∶1，得含药连接子MWD-L7为黄色固体67mg，收率23.1％，LC-MS(ESI)：M+1＝1682，(M+1)/2＝841。BL linker compound (146mg, 0.17mmol), HoSu (25mg, 0.21mmol, 1.2eq), DCC (54mg, 0.26mmol, 1.5eq) were dissolved in 20ml DCM, stirred at room temperature for 3h, and the reaction solution was suction filtered. The filtrate was added to the mixed solution of the crude intermediate 5-1, DIPEA (56mg, 0.43mmol, 2.5eq), and DCM (20ml), and the reaction was stirred for 3h. The reaction solution was cooled to 5-10°C, 1N hydrochloric acid (20ml) was added, and stirred for 0.5h. The layers were separated, the aqueous phase was extracted with DCM (30ml*2), and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered with suction, and spin-dried. Purified by column chromatography, eluent: DCM:MeOH=50:1～10:1, the drug-containing linker MWD-L7 was obtained as a yellow solid 67 mg, yield 23.1%, LC-MS (ESI): M+1= 1682, (M+1)/2=841.
实施例2.17含药连接子MWD-L8的合成 Example 2.17 Synthesis of drug-containing linker MWD-L8
Figure PCTCN2022139765-appb-000146
Figure PCTCN2022139765-appb-000146
根据实施例2.1类似合成方法，用
Figure PCTCN2022139765-appb-000147
代替BL化合物合成得到MWD-L8为淡黄色固体，LC-MS(ESI)：M+1＝1645.7。 According to the similar synthesis method of Example 2.1, use
Figure PCTCN2022139765-appb-000147
MWD-L8 was synthesized instead of BL compound as a pale yellow solid, LC-MS (ESI): M+1=1645.7.
实施例2.18含药连接子MWD-L9的合成 Example 2.18 Synthesis of drug-containing linker MWD-L9
Figure PCTCN2022139765-appb-000148
Figure PCTCN2022139765-appb-000148
根据实施例2.1类似合成方法，用
Figure PCTCN2022139765-appb-000149
代替BL化合物合成得到MWD-L9为淡黄色固体，LC-MS(ESI)：M+1＝1713.7。 According to the similar synthesis method of Example 2.1, use
Figure PCTCN2022139765-appb-000149
MWD-L9 was synthesized instead of BL compound as a pale yellow solid, LC-MS (ESI): M+1=1713.7.
实施例2.19含药连接子L-D的合成 Example 2.19 Synthesis of drug-containing linker LD
Figure PCTCN2022139765-appb-000150
Figure PCTCN2022139765-appb-000150
根据实施例2.5类似合成方法，用MC-OSU代替BL-OSU化合物合成得到L-D为淡黄色固体，LC-MS(ESI)：M+1＝795.9。According to the similar synthesis method of Example 2.5, MC-OSU was used instead of BL-OSU compound to synthesize L-D as light yellow solid, LC-MS (ESI): M+1=795.9.
实施例2.20含药连接子L-E的合成 Example 2.20 Synthesis of drug-containing linker LE
Figure PCTCN2022139765-appb-000151
Figure PCTCN2022139765-appb-000151
根据实施例2.2类似合成方法，用MC-OSU代替BL-OSU化合物合成得到L-E为淡黄色固体，LC-MS(ESI)：M+1＝803.8。According to the similar synthesis method of Example 2.2, MC-OSU was used instead of BL-OSU compound to synthesize L-E as light yellow solid, LC-MS (ESI): M+1=803.8.
实施例2.21含药连接子L-F的合成 Example 2.21 Synthesis of drug-containing linker LF
Figure PCTCN2022139765-appb-000152
Figure PCTCN2022139765-appb-000152
根据实施例2.2类似的方法，用A7代替A1，用MaL-PEG8-COOH代替BL化合物制备得到L-F，为黄色固体，LC-MS(ESI)：M+1＝1185。According to the similar method of Example 2.2, A7 was used to replace A1, and MaL-PEG8-COOH was used to replace BL compound to prepare L-F as a yellow solid, LC-MS (ESI): M+1=1185.
实施例2.22含药连接子L-G的合成 Example 2.22 Synthesis of drug-containing linker LG
Figure PCTCN2022139765-appb-000153
Figure PCTCN2022139765-appb-000153
根据实施例2.3，用MC-OSu化合物代替BL-OSu化合物，制备得到L-G，为黄色固体，MS(ESI)：M+1＝965.98。According to Example 2.3, MC-OSu compound was used instead of BL-OSu compound to prepare L-G as a yellow solid, MS (ESI): M+1=965.98.
实施例2.23含药连接子L-H的合成 Example 2.23 Synthesis of drug-containing linker LH
Figure PCTCN2022139765-appb-000154
Figure PCTCN2022139765-appb-000154
根据实施例2.4类似合成方法，用MaL-PEG8-COOH代替BL化合物合成得到L-H为淡黄色固体，LC-MS(ESI)：M+1＝1165.3。According to the similar synthesis method of Example 2.4, MaL-PEG8-COOH was used instead of BL compound to obtain L-H as light yellow solid, LC-MS (ESI): M+1=1165.3.
实施例2.24含药连接子L-I的合成 Example 2.24 Synthesis of drug-containing linker LI
Figure PCTCN2022139765-appb-000155
Figure PCTCN2022139765-appb-000155
用实施例2.2类似的方法，用A13代替A1，制备得到L-I，为黄色固体，LC-MS(ESI)：M+1＝1203.4。Using a method similar to Example 2.2, replacing A1 with A13, L-I was prepared as a yellow solid, LC-MS (ESI): M+1=1203.4.
实施例2.25含药连接子L-J的合成 Example 2.25 Synthesis of drug-containing linker LJ
Figure PCTCN2022139765-appb-000156
Figure PCTCN2022139765-appb-000156
用实施例2.2类似的方法，用A18代替A1，制备得到L-J，为黄色固体，LC-MS(ESI)：M+1＝1185.4。Using a method similar to Example 2.2, replacing A1 with A18, L-J was prepared as a yellow solid, LC-MS (ESI): M+1=1185.4.
实施例2.26含药连接子L-K的合成 Example 2.26 Synthesis of drug-containing linker LK
Figure PCTCN2022139765-appb-000157
Figure PCTCN2022139765-appb-000157
用实施例2.2类似的方法，用A16代替A1，制备得到L-K，为黄色固体，LC-MS(ESI)：M+1＝1169.3。Using a method similar to Example 2.2, replacing A1 with A16, L-K was prepared as a yellow solid, LC-MS (ESI): M+1=1169.3.
实施例2.27含药连接子L-L的合成 Example 2.27 Synthesis of drug-containing linker LL
Figure PCTCN2022139765-appb-000158
Figure PCTCN2022139765-appb-000158
用实施例2.2类似的方法，用A19代替A1，制备得到L-L，为黄色固体，LC-MS(ESI)：M+1＝1221.4。Using a method similar to Example 2.2, replacing A1 with A19, L-L was prepared as a yellow solid, LC-MS (ESI): M+1=1221.4.
实施例2.28含药连接子L-M的合成 Example 2.28 Synthesis of drug-containing linker LM
Figure PCTCN2022139765-appb-000159
Figure PCTCN2022139765-appb-000159
用实施例2.2的方法，用A17代替A1，制备得到L-M，为黄色固体，LC-MS(ESI)：M+1＝1205.4。Using the method of Example 2.2, replacing A1 with A17, L-M was prepared as a yellow solid, LC-MS (ESI): M+1=1205.4.
实施例2.29含药连接子L-N的合成 Example 2.29 Synthesis of drug-containing linker LN
Figure PCTCN2022139765-appb-000160
Figure PCTCN2022139765-appb-000160
按照实施例2.2类似的方法合成，用HCDE中间体代替CDE中间体；用MaL-PEG8-COOH代替BL化合物制备得到L-N，为黄色固体，LC-MS(ESI)：M+1＝1163.2(一对非对映异构体混合物)。Synthesize according to the method similar to Example 2.2, replace the CDE intermediate with the HCDE intermediate; replace the BL compound with MaL-PEG8-COOH to prepare L-N, which is a yellow solid, LC-MS (ESI): M+1=1163.2 (a pair diastereomeric mixture).
实施例2.30含药连接子MWS-L1的合成 Example 2.30 Synthesis of drug-containing linker MWS-L1
Figure PCTCN2022139765-appb-000161
Figure PCTCN2022139765-appb-000161
按照实施例2.2类似的方法用A13代替A1，用According to the similar method of embodiment 2.2, replace A1 with A13, use
Figure PCTCN2022139765-appb-000162
代替BL接头化合物制备得到MWS-L1，为黄色固体，LC-MS(ESI)：M+1＝1236.3。
Figure PCTCN2022139765-appb-000162
MWS-L1 was prepared instead of the BL linker compound as a yellow solid, LC-MS (ESI): M+1=1236.3.
实施例2.31含药连接子MWS-L2的合成 Example 2.31 Synthesis of drug-containing linker MWS-L2
Figure PCTCN2022139765-appb-000163
Figure PCTCN2022139765-appb-000163
用实施例2.2类似的方法，用A18代替A1，制备得到MWS-L2，为黄色固体，LC-MS(ESI)：M+1＝1218.4。Using a method similar to Example 2.2, replacing A1 with A18, MWS-L2 was prepared as a yellow solid, LC-MS (ESI): M+1=1218.4.
实施例2.32含药连接子MWS-L3的合成 Example 2.32 Synthesis of drug-containing linker MWS-L3
Figure PCTCN2022139765-appb-000164
Figure PCTCN2022139765-appb-000164
用实施例2.2类似的方法，用A16代替A1，制备得到MWS-L3，为黄色固体，LC-MS(ESI)：M+1＝1202.4。Using a method similar to Example 2.2, replacing A1 with A16, MWS-L3 was prepared as a yellow solid, LC-MS (ESI): M+1=1202.4.
实施例2.33含药连接子MWS-L4的合成 Example 2.33 Synthesis of drug-containing linker MWS-L4
Figure PCTCN2022139765-appb-000165
Figure PCTCN2022139765-appb-000165
用实施例2.2类似的方法，用A19代替A1，制备得到MWS-L4，为黄色固体，LC-MS(ESI)：M+1＝1254.4。Using a method similar to Example 2.2, replacing A1 with A19, MWS-L4 was prepared as a yellow solid, LC-MS (ESI): M+1=1254.4.
实施例2.34含药连接子MWS-L5的合成 Example 2.34 Synthesis of drug-containing linker MWS-L5
Figure PCTCN2022139765-appb-000166
Figure PCTCN2022139765-appb-000166
根据实施例2.2的方法，用A17代替A1，制备得到MWS-L5，为黄色固体，LC-MS(ESI)：M+1＝1238.4。According to the method of Example 2.2, A1 was replaced by A17 to prepare MWS-L5 as a yellow solid, LC-MS (ESI): M+1=1238.4.
实施例2.35含药连接子MWS-L6的合成 Example 2.35 Synthesis of drug-containing linker MWS-L6
Figure PCTCN2022139765-appb-000167
Figure PCTCN2022139765-appb-000167
按照实施例2.11类似的方法用According to the method similar to Example 2.11 with
Figure PCTCN2022139765-appb-000168
代替Mal-PEG8-COOH制备得到MWS-L6，为黄色固体，LC-MS(ESI)：M+1＝1302.4
Figure PCTCN2022139765-appb-000168
Instead of Mal-PEG8-COOH, MWS-L6 was prepared as a yellow solid, LC-MS (ESI): M+1=1302.4
实施例2.36含药连接子MWS-L7的合成 Example 2.36 Synthesis of drug-containing linker MWS-L7
Figure PCTCN2022139765-appb-000169
Figure PCTCN2022139765-appb-000169
用实施例2.2类似的方法，用A18代替A1，制备得到MWS-L7，为黄色固体，LC-MS(ESI)：M+1＝1284.4。Using a method similar to Example 2.2, replacing A1 with A18, MWS-L7 was prepared as a yellow solid, LC-MS (ESI): M+1=1284.4.
实施例2.37含药连接子MWS-L8的合成 Example 2.37 Synthesis of drug-containing linker MWS-L8
Figure PCTCN2022139765-appb-000170
Figure PCTCN2022139765-appb-000170
用实施例2.2类似的方法，用A16代替A1，制备得到MWS-L8，为黄色固体，LC-MS(ESI)：M+1＝1268.4。Using a method similar to Example 2.2, replacing A1 with A16, MWS-L8 was prepared as a yellow solid, LC-MS (ESI): M+1=1268.4.
实施例2.38含药连接子MWS-L9的合成 Example 2.38 Synthesis of drug-containing linker MWS-L9
Figure PCTCN2022139765-appb-000171
Figure PCTCN2022139765-appb-000171
用实施例2.2类似的方法，用A19代替A1，制备得到MWS-L9，为黄色固体，LC-MS(ESI)：M+1＝1320.4。Using a method similar to Example 2.2, replacing A1 with A19, MWS-L9 was prepared as a yellow solid, LC-MS (ESI): M+1=1320.4.
实施例2.39含药连接子MWS-L10的合成 Example 2.39 Synthesis of drug-containing linker MWS-L10
Figure PCTCN2022139765-appb-000172
Figure PCTCN2022139765-appb-000172
用实施例2.2类似的方法，用A17代替A1，制备得到MWS-L10，为黄色固体，LC-MS(ESI)：M+1＝1304.4。Using a method similar to Example 2.2, replacing A1 with A17, MWS-L10 was prepared as a yellow solid, LC-MS (ESI): M+1=1304.4.
实施例组3对比含药连接子的合成 Embodiment group 3 compares the synthesis of drug-containing linker
实施例3.1含药连接子L-A、L-B和L-C的合成 Example 3.1 Synthesis of drug-containing linkers LA, LB and LC
3.1.1随机偶联含药连接子的合成3.1.1 Synthesis of random coupling drug-containing linkers
含药连接子MC-GGFG-Dxd按照专利公开文件(US20190151328A1)所述方法合成，得到MC-GGFG-Dxd为淡黄色固体，LC-MS(ESI)：M+1＝1034， 1H NMR(CDCl 3)。 The drug-containing linker MC-GGFG-Dxd was synthesized according to the method described in the patent publication (US20190151328A1), and MC-GGFG-Dxd was obtained as a light yellow solid, LC-MS (ESI): M+1=1034, 1 H NMR (CDCl 3 ).
含药连接子L-A和L-B，按照专利公开文件(WO2020200880)所述方法合成，得到 L-A为黄色固体，LC-MS(ESI)：M+1＝767；L-B为棕色粘稠物，LC-MS(ESI)：M+1＝1149。Drug-containing linkers L-A and L-B were synthesized according to the method described in the patent publication (WO2020200880), and L-A was obtained as a yellow solid, LC-MS (ESI): M+1=767; L-B was a brown sticky substance, LC-MS ( ESI): M+1=1149.
Figure PCTCN2022139765-appb-000173
Figure PCTCN2022139765-appb-000173
3.2.2其他环桥连含药连接子L-C的合成3.2.2 Synthesis of other ring-bridged drug-containing linkers L-C
Figure PCTCN2022139765-appb-000174
Figure PCTCN2022139765-appb-000174
合成路线如下：The synthetic route is as follows:
Figure PCTCN2022139765-appb-000175
Figure PCTCN2022139765-appb-000175
按照含药连接子L-1类似的方法合成连接子L-C，L-C为淡黄色固体，LC-MS(ESI)：M+1＝1982，(M+1)/2＝991。Linker L-C was synthesized according to the method similar to drug-containing linker L-1. L-C was a pale yellow solid, LC-MS (ESI): M+1=1982, (M+1)/2=991.
实施例组4抗体药物偶联物的制备与理化表征 Example Group 4 Preparation and Physicochemical Characterization of Antibody Drug Conjugates
抗体药物偶联物制备工艺Antibody drug conjugate preparation process
a.定点偶联制备工艺a. Site-specific coupling preparation process
Figure PCTCN2022139765-appb-000176
Figure PCTCN2022139765-appb-000176
抗体经还原打开二硫键，再与连接子进行偶联形成桥接抗体药物偶联物，再经水解 打开马来酰亚胺环，最后经纯化得到DAR＝4的抗体-药物偶联物。The antibody is reduced to open the disulfide bond, then coupled with the linker to form a bridging antibody-drug conjugate, then hydrolyzed to open the maleimide ring, and finally purified to obtain an antibody-drug conjugate with DAR=4.
b.随机偶联制备工艺b. Random coupling preparation process
Figure PCTCN2022139765-appb-000177
Figure PCTCN2022139765-appb-000177
抗体经还原打开二硫键，再与连接子进行偶联形成桥接抗体药物偶联物。The antibody is reduced to open the disulfide bond, and then coupled with the linker to form a bridged antibody-drug conjugate.
示例性地，采用了以下实验方法：Exemplarily, the following experimental methods were used:
4.1抗体药物偶联物的通用制备方法4.1 General preparation method of antibody-drug conjugates
a.定点偶联的通用制备方法a. General preparation method for site-directed coupling
抗体的还原：将120mg的抗体样品使用Sephadex G25载体的NAP-25色谱柱、置换至pH值7.0含有50mM的氯化钠、50mM的磷酸二氢钠-磷酸氢二钠缓冲溶液中，并将抗体浓度稀释至10mg/ml。取10ml共计100mg的抗体样品，以抗体-TCEP摩尔比1比10当量添加10mg/ml的TCEP(Sigma-Aldrich)的水溶液，加入TCEP水溶液的体积为2.1ml。孵育2小时后，使用Sephadex G25色谱柱反应溶液进行置换，置换至pH 6.5含有50mM氯化钠，50mM磷酸二氢钠-磷酸氢二钠缓冲的缓冲溶液中。Antibody reduction: use Sephadex G25 carrier NAP-25 chromatographic column to replace 120mg antibody sample to pH 7.0 containing 50mM sodium chloride, 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, and the antibody The concentration was diluted to 10mg/ml. Take 10ml of antibody samples totaling 100mg, and add 10mg/ml TCEP (Sigma-Aldrich) aqueous solution at an antibody-TCEP molar ratio of 1:10 equivalent, and the volume of TCEP aqueous solution added is 2.1ml. After 2 hours of incubation, use the Sephadex G25 chromatographic column reaction solution to replace to a pH 6.5 buffer solution containing 50mM sodium chloride, 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer.
抗体与含药连接子的偶联及水解：将上述还原后的抗体稀释至5mg/mL，依次加入反应总体积2％的0.38ml N，N-二甲基乙酰胺(DMA)0.38ml作为预溶剂，以抗体-小分子药物摩尔比1比5.5倍当量添加0.67ml含10mg/mL含药连接子的DMA-含药连接子混合溶液作为反应液。室温搅拌30分钟。使用Sephadex G25载体的NAP-25色谱柱将反应液置换至pH8.0的磷酸氢二钠-磷酸二氢钠缓冲溶液，除去过量的含药连接子，37℃水浴加热3小时。Coupling and hydrolysis of antibody and drug-containing linker: Dilute the above-mentioned reduced antibody to 5mg/mL, and add 0.38ml of N, N-dimethylacetamide (DMA) 0.38ml of 2% of the total reaction volume in turn as a prepreparation solution. As a solvent, add 0.67 ml of DMA-drug-containing linker mixed solution containing 10 mg/mL drug-containing linker at an antibody-small molecule drug molar ratio of 1:5.5 equivalents as a reaction solution. Stir at room temperature for 30 minutes. The NAP-25 chromatographic column with Sephadex G25 carrier was used to replace the reaction solution to the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH 8.0 to remove the excess drug-containing linker, and heated in a water bath at 37°C for 3 hours.
抗体药物偶联物的纯化：将上述样品进行使用AMICOM超滤离心管进行浓缩，将样品浓缩至15mg/mL左右。添加50mM磷酸氢二钠-磷酸二氢钠+3M磷酸铵缓冲溶液至电导率为100ms/cm。上样至TOYOPEAL Butyl-650M填料(购自TOSOH)疏水柱，A相为50mm磷酸氢二钠-磷酸二氢钠+0.6M硫酸铵，B相为50mm磷酸氢二钠-磷酸二氢钠的缓冲溶液。B相0-100％梯度8倍柱体积洗脱，对主峰进行收集。Purification of antibody-drug conjugates: Concentrate the above samples using AMICOM ultrafiltration centrifuge tubes, and concentrate the samples to about 15 mg/mL. Add 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate+3M ammonium phosphate buffer solution until the conductivity is 100ms/cm. Load the sample to TOYOPEAL Butyl-650M filler (purchased from TOSOH) hydrophobic column, phase A is 50mm disodium hydrogen phosphate-sodium dihydrogen phosphate + 0.6M ammonium sulfate, phase B is buffered with 50mm disodium hydrogen phosphate-sodium dihydrogen phosphate solution. Phase B was eluted with a 0-100% gradient of 8 column volumes, and the main peak was collected.
使用AMICOM超滤离心管将最终样品置换至pH值7.4的50mM的磷酸氢二钠-磷酸二氢钠缓冲盐中，并使用0.22um的滤膜进行过滤(Sartorius stedim Ministart)。Use an AMICOM ultrafiltration centrifuge tube to replace the final sample into 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer salt with a pH value of 7.4, and filter with a 0.22um filter membrane (Sartorius stedim Ministart).
b.随机偶联的通用制备方法b. General Preparation Method for Random Coupling
按照CN 105849126 A专利公开的制备方法制备得到。Prepared according to the preparation method disclosed in CN 105849126 A patent.
4.2抗体药物偶联物的通用分析方法4.2 General analytical methods for antibody drug conjugates
a.紫外分光光度法测定药物抗体偶联比(UV-DAR法)与浓度a. Determination of drug-antibody coupling ratio (UV-DAR method) and concentration by ultraviolet spectrophotometry
抗体药物偶联物浓度可通过测定在280nm及小分子特征吸收波长下的UV吸光度，然后进行下述计算而得到。The concentration of the antibody-drug conjugate can be obtained by measuring the UV absorbance at 280 nm and the characteristic absorption wavelength of small molecules, and then performing the following calculations.
a1.抗体药物偶联物的药物抗体偶联比(DAR)测定a1. Determination of Drug Antibody Conjugation Ratio (DAR) of Antibody Drug Conjugates
根据文献[Clin Cancer Res.2004 Oct 15；10(20)：7063-70]可知
Figure PCTCN2022139765-appb-000178
Figure PCTCN2022139765-appb-000179
其中，
Figure PCTCN2022139765-appb-000180
为抗体在280nm下摩尔吸收系数，A 280为抗体药物偶联物在280nm下紫外吸收值，A z为抗体药物偶联物在含药连接子特征吸收波长Z nm下紫外吸收值，
Figure PCTCN2022139765-appb-000181
为抗体在含药连接子特征吸收波长Z nm下摩尔吸收系数，
Figure PCTCN2022139765-appb-000182
为含药连接子在其特征吸收波长Z nm下摩尔吸收系数，
Figure PCTCN2022139765-appb-000183
为含药连接子在280nm下摩尔吸收系数。 According to the literature [Clin Cancer Res.2004 Oct 15; 10(20): 7063-70]
Figure PCTCN2022139765-appb-000178
Figure PCTCN2022139765-appb-000179
in,
Figure PCTCN2022139765-appb-000180
is the molar absorption coefficient of the antibody at 280nm, A 280 is the ultraviolet absorption value of the antibody-drug conjugate at 280nm, Az is the ultraviolet absorption value of the antibody-drug conjugate at the characteristic absorption wavelength Z nm of the drug-containing linker,
Figure PCTCN2022139765-appb-000181
is the molar absorption coefficient of the antibody at the characteristic absorption wavelength Z nm of the drug-containing linker,
Figure PCTCN2022139765-appb-000182
is the molar absorption coefficient of the drug-containing linker at its characteristic absorption wavelength Z nm,
Figure PCTCN2022139765-appb-000183
is the molar absorption coefficient of the drug-containing linker at 280 nm.
其中：in:
Figure PCTCN2022139765-appb-000184
Figure PCTCN2022139765-appb-000184
a2.药物浓度测定a2. Determination of drug concentration
由于某一波长下的总吸光度等于存在于体系内的所有吸收化学物质种类的吸光度的和(吸光度的加成性)，所以假设在抗体与含药连接子偶联前后，抗体及含药连接子的摩尔吸光系数不发生变化时，抗体药物偶联物浓度有如下关系：Since the total absorbance at a certain wavelength is equal to the sum of the absorbances of all types of absorbing chemical species present in the system (additive absorbance), it is assumed that the antibody and the drug-containing linker will When the molar absorptivity does not change, the concentration of the antibody-drug conjugate has the following relationship:
Figure PCTCN2022139765-appb-000185
Figure PCTCN2022139765-appb-000185
因此，抗体药物偶联物摩尔浓度(mol/L)
Figure PCTCN2022139765-appb-000186
由此得到抗体药物偶联物浓度(g/L)
Figure PCTCN2022139765-appb-000187
Figure PCTCN2022139765-appb-000188
将DAR值代入式中即得蛋白浓度。 Therefore, the antibody drug conjugate molar concentration (mol/L)
Figure PCTCN2022139765-appb-000186
Thus the concentration of antibody drug conjugate (g/L) was obtained
Figure PCTCN2022139765-appb-000187
Figure PCTCN2022139765-appb-000188
Substitute the DAR value into the formula to obtain the protein concentration.
其中，MW ADC为抗体药物偶联物分子量，MW Ab为抗体分子量，MW D为含药连接子分子量。 Wherein, MW ADC is the molecular weight of the antibody-drug conjugate, MW Ab is the molecular weight of the antibody, and MW D is the molecular weight of the drug-containing linker.
b.疏水层析法b. Hydrophobic chromatography
b1.抗体药物偶联物疏水色谱HIC-HPLC测定DAR值b1. Determination of DAR value of antibody-drug conjugates by HIC-HPLC
样品制备：样品用流动相B稀释至2.0mg/ml后，12000rpm离心10min，取上清用于HPLC分析；Sample preparation: Dilute the sample to 2.0mg/ml with mobile phase B, centrifuge at 12000rpm for 10min, and take the supernatant for HPLC analysis;
色谱柱：Sepax Proteomix HIC Butyl-NP5，5μm，4.6mm*35mm；Chromatographic column: Sepax Proteomix HIC Butyl-NP5, 5μm, 4.6mm*35mm;
流动相A：1.5M(NH4) 2SO 4+25mM PB，pH 7.0； Mobile phase A: 1.5M (NH4) 2 SO 4 +25mM PB, pH 7.0;
流动相B：25mM PB+20％IPA，pH 7.0；Mobile phase B: 25mM PB+20%IPA, pH 7.0;
流速：0.6mL/min；Flow rate: 0.6mL/min;
检测波长：280nm；Detection wavelength: 280nm;
柱温：30℃；Column temperature: 30°C;
上样量：10μL；Loading volume: 10 μL;
HIC色谱梯度HIC chromatographic gradient
时间time ％A%A ％B%B
00 100100 00
1010 8080 2020
2020 4040 6060
2525 00 100100
25.125.1 100100 00
3535 100100 00
DAR计算公式：DAR calculation formula:
DAR＝∑(加权峰面积)/100，即DAR＝(D0峰面积比*0+D1峰面积比*1+D2峰面积比*2+D3峰面积比*3+D4峰面积比*4+D5峰面积比*5+D6峰面积比*6+D7峰面积比*7+D8峰面积比*8)/100。DAR=∑(weighted peak area)/100, namely DAR=(D0 peak area ratio*0+D1 peak area ratio*1+D2 peak area ratio*2+D3 peak area ratio*3+D4 peak area ratio*4+ D5 peak area ratio*5+D6 peak area ratio*6+D7 peak area ratio*7+D8 peak area ratio*8)/100.
b2.抗体药物偶联物疏水色谱HIC-HPLC测定DAR值b2. Determination of DAR value of antibody-drug conjugates by HIC-HPLC
样品制备：样品用流动相B稀释至2.0mg/ml后，12000rpm离心10min，取上清用于HPLC分析；Sample preparation: Dilute the sample to 2.0mg/ml with mobile phase B, centrifuge at 12000rpm for 10min, and take the supernatant for HPLC analysis;
色谱柱：TSKgel Butyl-NPR，2.5μm，4.6mm*100mm；Chromatographic column: TSKgel Butyl-NPR, 2.5μm, 4.6mm*100mm;
流动相A：1.2M(NH4) 2SO 4+25mM PB，pH 7.0； Mobile phase A: 1.2M (NH4) 2 SO 4 +25mM PB, pH 7.0;
流动相B：25mM PB+20％IPA，pH 7.0；Mobile phase B: 25mM PB+20%IPA, pH 7.0;
流速：0.6mL/min；Flow rate: 0.6mL/min;
检测波长：280nm；Detection wavelength: 280nm;
柱温：30℃；Column temperature: 30°C;
上样量：10μL；Loading volume: 10 μL;
HIC色谱梯度HIC chromatographic gradient
时间time ％A%A ％B%B
00 100100 00
11 100100 00
2020 00 100100
20.120.1 100100 00
3030 100100 00
DAR计算公式：同b1。DAR calculation formula: same as b1.
b3.抗体药物偶联物疏水色谱HIC-HPLC测定DAR值b3. Determination of DAR value of antibody-drug conjugates by HIC-HPLC
样品制备：样品用流动相B稀释至2.0mg/ml后，12000rpm离心10min，取上清用于HPLC分析；Sample preparation: Dilute the sample to 2.0mg/ml with mobile phase B, centrifuge at 12000rpm for 10min, and take the supernatant for HPLC analysis;
色谱柱：Sepax Proteomix HIC Butyl-NP5，5μm，4.6mm*35mm；Chromatographic column: Sepax Proteomix HIC Butyl-NP5, 5μm, 4.6mm*35mm;
流动相A：2.5M(NH4) 2SO 4+125mM PB，pH 7.0 Mobile phase A: 2.5M (NH4) 2 SO 4 +125mM PB, pH 7.0
流动相B：125mM PB，pH 7.0Mobile Phase B: 125mM PB, pH 7.0
流速：0.5mL/min；Flow rate: 0.5mL/min;
检测波长：280nm；Detection wavelength: 280nm;
柱温：30℃；Column temperature: 30°C;
上样量：10μL；Loading volume: 10 μL;
HIC色谱梯度HIC chromatographic gradient
时间time ％A%A ％B%B ％C%C ％D%D
00 8080 00 00 2020
2020 55 4545 33 4747
22twenty two 00 5050 33 4747
22.122.1 8080 00 00 2020
3030 8080 00 00 2020
DAR计算公式：同b1。DAR calculation formula: same as b1.
c.质谱法LC-MS测定DAR值c. Determination of DAR value by mass spectrometry LC-MS
样品处理：取适量样品于超滤管中，采用50mM NH4HCO3置换缓冲液(pH7.1)进行置换，补加缓冲液，超滤离心(13000g*5min)。加入8μl的PNGase F酶至置换后的样品中，37℃孵育5h进行脱糖处理，孵育结束后，12000rpm离心5min，取上清液于进样小瓶中作为供试样品待用。Sample treatment: Take an appropriate amount of sample in an ultrafiltration tube, replace it with 50mM NH4HCO3 replacement buffer (pH7.1), add buffer, and ultrafiltration centrifuge (13000g*5min). Add 8 μl of PNGase F enzyme to the replaced sample, incubate at 37°C for 5 hours for desugaring treatment, after the incubation, centrifuge at 12000rpm for 5 minutes, take the supernatant into the injection vial as the test sample for use.
色谱柱：
Figure PCTCN2022139765-appb-000189
PolyHYDROXYETHYLA Column，
Figure PCTCN2022139765-appb-000190
5um.2.1mm×200mm； Column:
Figure PCTCN2022139765-appb-000189
PolyHYDROXYETHYLA Column,
Figure PCTCN2022139765-appb-000190
5um.2.1mm×200mm;
流动相：50mM乙酸铵，pH 7.0；Mobile phase: 50mM ammonium acetate, pH 7.0;
运行时间：10min；Running time: 10min;
流速：0.1mL/min；Flow rate: 0.1mL/min;
进样体积：2μL；Injection volume: 2μL;
柱温：25℃；Column temperature: 25°C;
检测波长：280nm；Detection wavelength: 280nm;
离子化方式：ESI positiveIonization method: ESI positive
干燥气体温度：325℃Drying gas temperature: 325°C
干燥气体流速：8L/minDrying gas flow rate: 8L/min
雾化器压力：20psigAtomizer pressure: 20psig
鞘气温度：325℃；Sheath gas temperature: 325°C;
鞘气流量：12L/min；Sheath gas flow: 12L/min;
扫描设置：900-8000m/z。Scan settings: 900-8000m/z.
d.分子排阻色谱SEC-HPLC测定分子大小异质性d. Molecular Size Heterogeneity Determination by Size Exclusion Chromatography SEC-HPLC
样品处理：样品用流动相稀释至约1.0mg/ml后，12000rpm离心10min，取上清进样分析。Sample processing: After diluting the sample to about 1.0 mg/ml with mobile phase, centrifuge at 12000 rpm for 10 min, and take the supernatant for sample analysis.
色谱柱：TOSOH，TSKgel G3000SWXL，5μm，7.8mm*300mm；Chromatographic column: TOSOH, TSKgel G3000SWXL, 5μm, 7.8mm*300mm;
流动相：100mM PB+200mM盐酸精氨酸，5％异丙醇(pH 6.8)；Mobile phase: 100mM PB+200mM arginine hydrochloride, 5% isopropanol (pH 6.8);
流速：0.6mL/min；Flow rate: 0.6mL/min;
检测波长：280nm；Detection wavelength: 280nm;
柱温：30℃；Column temperature: 30°C;
上样量：20uL；Sample volume: 20uL;
洗脱时间：20min；Elution time: 20min;
洗脱梯度：等度洗脱。Elution gradient: isocratic elution.
e.非还原毛细管电泳NR-CE-SDS测定纯度e. Determination of purity by non-reducing capillary electrophoresis NR-CE-SDS
根据《中国药典四部》通则3127单抗分子大小变异体测定法测定。According to the "Chinese Pharmacopoeia IV" general rule 3127 monoclonal antibody molecular size variant determination method.
f.反向作用色谱(RP-HPLC)法测定抗体药物偶联物小分子/抗体偶联比(DAR值)f. Determination of Antibody-drug Conjugate Small Molecule/Antibody Conjugation Ratio (DAR Value) by Reverse Action Chromatography (RP-HPLC)
按照专利公开文件(US 10227417B2)所述方法分析。Analyze according to the method described in patent publication (US 10227417B2).
根据公式计算DAR值：DAR＝2×(∑轻链加权峰面积+∑重链加权峰面积)/100。即：DAR＝2×(L0峰面积比×0+L1峰面积比×1+H0峰面积比×0+H1峰面积比×1+H2峰面积比×2+H3峰面积比×3)/100。The DAR value was calculated according to the formula: DAR=2×(Σ light chain weighted peak area+Σ heavy chain weighted peak area)/100. That is: DAR=2×(L0 peak area ratio×0+L1 peak area ratio×1+H0 peak area ratio×0+H1 peak area ratio×1+H2 peak area ratio×2+H3 peak area ratio×3)/ 100.
g.反向作用色谱(RP-HPLC)法测定抗体药物偶联物小分子/抗体偶联比(DAR值)g. Determination of antibody-drug conjugate small molecule/antibody conjugation ratio (DAR value) by reverse action chromatography (RP-HPLC)
样品前处理：按照专利公开文件(US 10227417 B2)所述方法处理样品。Sample pretreatment: Process the sample according to the method described in the patent publication (US 10227417 B2).
HPLC仪器：Waters Acquity ArcHPLC instrument: Waters Acquity Arc
流动相A：0.1％TFA+ACNMobile phase A: 0.1% TFA+ACN
流动相B：0.1％TFA+H2OMobile phase B: 0.1% TFA+H2O
分析柱：Waters；BioResolve；2.1*100mm；2.7μm；
Figure PCTCN2022139765-appb-000191
PN 0024168 Analytical column: Waters; BioResolve; 2.1*100mm; 2.7μm;
Figure PCTCN2022139765-appb-000191
PN 0024168
进样体积：5μLInjection volume: 5μL
流速：0.25mL/minFlow rate: 0.25mL/min
柱温：80℃Column temperature: 80°C
检测器：PDA检测器Detector: PDA detector
检测波长：280nmDetection wavelength: 280nm
梯度：gradient:
时间(min)time (min) 流动相Amobile phase A 流动相B mobile phase B
00 2525 7575
12.512.5 3636 6464
2020 4545 5555
2525 4545 5555
25.125.1 2525 7575
3535 2525 7575
实施例4.1靶向Trop-2的抗体药物偶联物的制备与表征 Example 4.1 Preparation and Characterization of Antibody-Drug Conjugates Targeting Trop-2
根据本实施例组4的偶联方法，将靶向Trop-2抗体hTINA1(根据WHO Drug Information中Datopotamab的序列进行制备)、h23-12各100mg，分别与含药连接子MWD-L1、MWC-L2、MWC-L3、MWF-L6、MWD-L7、MWD-L8、MWD-L9及MWF-L8制备ADC。得到桥连ADC药物1a、1b、1c、1d、1j、1k、1l、1m、1n、1o和1p；收集1d 未进行疏水层析的中间样品，作为中间样品1j保存。According to the coupling method of group 4 of this example, 100 mg each of the targeting Trop-2 antibody hTINA1 (prepared according to the sequence of Datopotamab in WHO Drug Information) and h23-12 were respectively combined with drug-containing linkers MWD-L1 and MWC- ADCs were prepared from L2, MWC-L3, MWF-L6, MWD-L7, MWD-L8, MWD-L9 and MWF-L8. The bridged ADC drugs 1a, 1b, 1c, 1d, 1j, 1k, 1l, 1m, 1n, 1o, and 1p were obtained; the intermediate samples of 1d without hydrophobic chromatography were collected and saved as intermediate sample 1j.
根据专利CN105849126B方法，将靶向Trop-2抗体hTINA1、h23-12各100mg，分别与MC-GGFG-Dxd、L-A、L-B、L-J、MWS-L7、L-I及MWS-L6制备ADC，得到随机偶联ADC1e、1f、1g、1h、1i、1q、1r、1s、1t和1u。According to the method of patent CN105849126B, 100mg each of the targeting Trop-2 antibodies hTINA1 and h23-12 were mixed with MC-GGFG-Dxd, L-A, L-B, L-J, MWS-L7, L-I and MWS-L6 respectively to prepare ADCs to obtain random coupling ADC 1e, 1f, 1g, 1h, 1i, 1q, 1r, 1s, 1t, and 1u.
使用本实施例组4中紫外分光光度法4.2a、疏水层析法4.2b以及分子排阻色谱法4.2d分别对桥连ADC(1a-1d、1j、1k-1p)的DAR值、浓度及纯度进行测定。The DAR value, concentration and Purity was measured.
使用本实施例组4中反向作用色谱法4.2f对含MC-GGFG-Dxd含药连接子的ADC样品(1e、1h、1q、1r、1s、1t及1u)DAR值进行测定、反向色谱法4.2g对含L-A或L-B含药连接子的ADC样品(1f、1g、1i)DAR值进行测定；使用实施例组4中4.2a紫外分光光度法以及4.2d分子排阻色谱法分别对非桥连ADC药物(1e-1i)的浓度及纯度进行测定。Use reverse action chromatography 4.2f in group 4 of this embodiment to measure the DAR value of ADC samples (1e, 1h, 1q, 1r, 1s, 1t and 1u) containing MC-GGFG-Dxd drug-containing linker, reverse Chromatography 4.2g measures the DAR value of ADC samples (1f, 1g, 1i) containing L-A or L-B drug-containing linkers; use 4.2a ultraviolet spectrophotometry and 4.2d size exclusion chromatography in Example Group 4 to determine The concentration and purity of non-bridging ADC drugs (1e-1i) were determined.
结果见表1。The results are shown in Table 1.
表1.靶向Trop-2的抗体药物偶联物的表征结果Table 1. Characterization results of antibody-drug conjugates targeting Trop-2
Figure PCTCN2022139765-appb-000192
Figure PCTCN2022139765-appb-000192
Figure PCTCN2022139765-appb-000193
Figure PCTCN2022139765-appb-000193
(本发明中，h23-12为具有如下序列的单克隆抗体：(In the present invention, h23-12 is a monoclonal antibody with the following sequence:
重链可变区：Heavy chain variable region:
Figure PCTCN2022139765-appb-000194
Figure PCTCN2022139765-appb-000194
重链CDR1：SYWMH(SEQ ID NO：1)Heavy chain CDR1: SYWMH (SEQ ID NO: 1)
重链CDR2：EITPSDNYGSYNQKFKG(SEQ ID NO：2)Heavy chain CDR2: EITPSDNYGSYNQKFKG (SEQ ID NO: 2)
重链CDR3：GHGNYVSFDY(SEQ ID NO：3)Heavy chain CDR3: GHGNYVSFDY (SEQ ID NO: 3)
轻链可变区：Light chain variable region:
Figure PCTCN2022139765-appb-000195
Figure PCTCN2022139765-appb-000195
轻链CDR1氨基酸：RASQDISNYLN(SEQ ID NO：4)Light chain CDR1 amino acid: RASQDISNYLN (SEQ ID NO: 4)
轻链CDR2氨基酸：YTSRLES(SEQ ID NO：5)Light chain CDR2 amino acid: YTSRLES (SEQ ID NO: 5)
轻链CDR3氨基酸：QQGYTLPPYT(SEQ ID NO：6)Light chain CDR3 amino acid: QQGYTLPPYT (SEQ ID NO: 6)
重链恒定区：Heavy chain constant region:
Figure PCTCN2022139765-appb-000196
Figure PCTCN2022139765-appb-000196
轻链恒定区：Light chain constant region:
Figure PCTCN2022139765-appb-000197
Figure PCTCN2022139765-appb-000197
实施例4.2靶向Her2抗体药物偶联物的制备 Example 4.2 Preparation of Drug Conjugates Targeting Her2 Antibody
根据本实施例组4的偶联方法，将靶向HER-2抗体曲妥珠单抗(CAS：180288-69-1， 购自上海罗氏制药有限公司)各100mg，分别与含药连接子MWD-L1、MWD-L2、MWE-L4、MWF-L6、MWD-L8、MWD-L9、L-C，MWC-L3进行制备桥连ADC 2a、2b、2c、2h、2i、2d，2l。According to the conjugation method of group 4 of this example, 100 mg of the HER-2-targeting antibody trastuzumab (CAS: 180288-69-1, purchased from Shanghai Roche Pharmaceutical Co., Ltd.) was mixed with the drug-containing linker MWD -L1, MWD-L2, MWE-L4, MWF-L6, MWD-L8, MWD-L9, L-C, MWC-L3 were used to prepare bridging ADC 2a, 2b, 2c, 2h, 2i, 2d, 2l.
分别根据专利CN105829346B方法，将靶向HER-2抗体曲妥珠单抗各100mg分别与MC-GGFG-DXD、L-A、L-B制备随机偶联ADC 2e，2f，2g，2k，2m。According to the patent CN105829346B method, 100 mg of the HER-2-targeting antibody trastuzumab was mixed with MC-GGFG-DXD, L-A, and L-B to prepare random conjugated ADCs 2e, 2f, 2g, 2k, and 2m.
使用本实施例组4中紫外分光光度法4.2a、疏水层析法4.2b以及分子排阻色谱法4.2d分别对桥连ADC(2a-2d，2h-2j，2l)的DAR值、浓度及纯度进行测定。The DAR value, concentration and Purity was measured.
使用实施例组4中反向色谱法4.2f对含MC-GGFG-Dxd含药连接子的ADC样品(2g)DAR值进行测定、反向色谱法4.2g对含L-A或L-B含药连接子的ADC样品(2e、2b)DAR值进行测定；使用紫外分光光度法4.2a以及分子排阻色谱法4.2d分别对非桥连ADC药物(2e-2g，2k，2m)的DAR值、浓度及纯度进行测定。Use reverse chromatography 4.2f in embodiment group 4 to measure the ADC sample (2g) DAR value containing MC-GGFG-Dxd drug-containing linker, reverse chromatography 4.2g to contain L-A or L-B drug-containing linker The DAR value of the ADC sample (2e, 2b) was determined; the DAR value, concentration and purity of the non-bridging ADC drug (2e-2g, 2k, 2m) were determined by UV spectrophotometry 4.2a and molecular exclusion chromatography 4.2d respectively To measure.
结果见表2。The results are shown in Table 2.
表2.靶向HER-2的抗体药物偶联物的表征结果Table 2. Characterization results of antibody-drug conjugates targeting HER-2
序号serial number 名称name 抗体Antibody 含药连接子drug-containing linker DARDAR 浓度mg/mlConcentrationmg/ml SECSEC
2a2a Her2-ADC-1Her2-ADC-1 曲妥珠单抗Trastuzumab MWC-L1MWC-L1 3.93.9 4.64.6 98.0％98.0%
2b2b Her2-ADC-2Her2-ADC-2 曲妥珠单抗Trastuzumab MWC-L2MWC-L2 4.04.0 3.83.8 99.2％99.2%
2c2c Her2-ADC-3Her2-ADC-3 曲妥珠单抗Trastuzumab MWE-L4MWE-L4 4.04.0 3.73.7 97.5％97.5%
2d2d Her2-ADC-4Her2-ADC-4 曲妥珠单抗Trastuzumab L-CL-C 3.93.9 3.83.8 97.7297.72
2e2e Her2-ADC-5Her2-ADC-5 曲妥珠单抗Trastuzumab L-AL-A 4.34.3 4.84.8 99.0％99.0%
2f2f Her2-ADC-6Her2-ADC-6 曲妥珠单抗Trastuzumab L-BL-B 4.14.1 6.26.2 98.6％98.6%
2g2g Her2-ADC-7Her2-ADC-7 曲妥珠单抗Trastuzumab MC-GGFG-DxdMC-GGFG-Dxd 4.04.0 6.66.6 99.8％99.8%
2h2 hours Her2-ADC-8Her2-ADC-8 曲妥珠单抗Trastuzumab MWD-L8MWD-L8 4.04.0 7.57.5 98.9％98.9%
2i2i Her2-ADC-9Her2-ADC-9 曲妥珠单抗Trastuzumab MWD-L9MWD-L9 4.04.0 7.87.8 99.5％99.5%
2j2j Her2-ADC-10Her2-ADC-10 曲妥珠单抗Trastuzumab MWF-L6MWF-L6 4.04.0 7.27.2 98.7％98.7%
2k2k Her2-ADC-11Her2-ADC-11 曲妥珠单抗Trastuzumab MC-GGFG-DxdMC-GGFG-Dxd 8.08.0 5.35.3 99.2％99.2%
2l2l Her2-ADC-12Her2-ADC-12 曲妥珠单抗Trastuzumab MWC-L3MWC-L3 3.53.5 16.716.7 99.5％99.5%
2m2m Her2-ADC-13Her2-ADC-13 曲妥珠单抗Trastuzumab L-BL-B 8.08.0 1.81.8 95.0％95.0%
实施例4.3化合物的结构表征 Example 4.3 Structural characterization of compounds
对实施例1和实施例2制备的Trop2样品(1a、1h、1j)以及Her2样品(2b、2e)如下分别使用本实施例组4中4.2b进行疏水层析分析，以比较不同连接技术产生的ADC的药物 异质性。The Trop2 samples (1a, 1h, 1j) and Her2 samples (2b, 2e) prepared in Example 1 and Example 2 were analyzed by hydrophobic chromatography using 4.2b in Group 4 of this example as follows to compare the results of different connection techniques. Drug Heterogeneity of ADCs.
结果见表3。The results are shown in Table 3.
表3.抗体药物偶联物的表征结果Table 3. Characterization results of antibody drug conjugates
序号serial number 名称name 抗体Antibody 含药连接子drug-containing linker HIC层析方法HIC chromatography method
1d1d Trop2-ADC-4Trop2-ADC-4 hTINA1hTINA1 MWC-L1MWC-L1 4.2.b34.2.b3
1h1h Trop2-ADC-8Trop2-ADC-8 hTINA1hTINA1 MC-GGFG-DxdMC-GGFG-Dxd 4.2.b24.2.b2
1j1j Trop2-ADC-4’Trop2-ADC-4' hTINA1hTINA1 MWC-L1MWC-L1 4.2.b34.2.b3
2b2b Her2-ADC-2Her2-ADC-2 曲妥珠单抗Trastuzumab MWC-L2MWC-L2 4.2.b34.2.b3
2e2e Her2-ADC-5Her2-ADC-5 曲妥珠单抗Trastuzumab L-AL-A 4.2.b24.2.b2
比较Trop2-ADC-4’、Trop2-ADC-4以及Trop2-ADC-8可知，使用含药连接子MWD-L1制备的ADC药物Trop2-ADC-4和Trop2-ADC-4’与使用MC-GGFG-Dxd制备的已知ADC药物(Trop2-ADC-8)相比，具有更优异的药物均一性。结果见图1。Comparing Trop2-ADC-4', Trop2-ADC-4 and Trop2-ADC-8, it can be seen that the ADC drugs Trop2-ADC-4 and Trop2-ADC-4' prepared using the drug-containing linker MWD-L1 were compared with those prepared using MC-GGFG Compared with the known ADC drug (Trop2-ADC-8) prepared by -Dxd, it has more excellent drug uniformity. The results are shown in Figure 1.
比较Her2-ADC-2以及Her2-ADC-5可知，使用含药连接子MWC-L2制备的ADC药物Her2-ADC-2比使用已知连接子L-A制备的Her2-ADC-5的均一性更佳。Comparing Her2-ADC-2 and Her2-ADC-5, it can be seen that the ADC drug Her2-ADC-2 prepared using the drug-containing linker MWC-L2 has better uniformity than Her2-ADC-5 prepared using the known linker L-A .
实施例组5测活评价 Example group 5 biopsy evaluation
实施例5.1喜树碱类化合物活性对比研究 Example 5.1 Comparative Study on the Activity of Camptothecin Compounds
口腔上皮癌细胞KB(购自：ATCC)使用DMEM培养基添加10％FBS培养；人胃癌细胞NCI-N87(购自：ATCC)使用RPMI1640培养基添加10％FBS培养；人结肠癌细胞HT29(购自：ATCC)使用RPMI1640培养基添加10％FBS培养；人胰腺腺癌细胞BxPC-3(购自：ATCC)使用RPMI1640培养基添加10％FBS培养。Oral epithelial cancer cells KB (purchased from: ATCC) were cultured in DMEM medium supplemented with 10% FBS; human gastric cancer cells NCI-N87 (purchased from: ATCC) were cultured in RPMI1640 medium supplemented with 10% FBS; human colon cancer cells HT29 (purchased from: From: ATCC) was cultured using RPMI1640 medium supplemented with 10% FBS; human pancreatic adenocarcinoma cell BxPC-3 (purchased from: ATCC) was cultured using RPMI1640 medium supplemented with 10% FBS.
使用上述相应培养基将KB细胞调整细胞密度至2×10 4个/mL；将NCI-N87细胞调整细胞密度至5×10 4个/mL；将HT29细胞调整细胞密度至5×10 4个/mL，100μL/孔铺于96孔透明平底板中，过夜培养；将BxPC-3细胞调整细胞密度至5×10 4个/mL。 Use the above-mentioned corresponding medium to adjust the cell density of KB cells to 2×10 4 cells/mL; adjust the cell density of NCI-N87 cells to 5×10 4 cells/mL; adjust the cell density of HT29 cells to 5×10 4 cells/mL mL, 100 μL/well was spread in a 96-well transparent flat-bottom plate, and cultured overnight; the BxPC-3 cells were adjusted to a cell density of 5×10 4 cells/mL.
使用培养基稀释待测样品至20μM，然后5倍稀释，设置9个梯度(设置不加样品孔即细胞最大生长孔和不加细胞孔即培养基本底)。按100μL/孔加入细胞培养板中，二氧化碳培养箱孵育96小时。孵育完毕后，按照20μL/孔加入配置好的CCK-8(购自：同仁化学)，二氧化碳培养箱孵育3小时后，酶标仪读取OD450值。Use culture medium to dilute the sample to be tested to 20 μM, then 5-fold dilution, and set 9 gradients (set no sample well, which is the maximum cell growth well and no cell well, which is the basic culture base). Add 100 μL/well to the cell culture plate, and incubate for 96 hours in a carbon dioxide incubator. After the incubation, the prepared CCK-8 (purchased from Tongren Chemical) was added at 20 μL/well, and after incubation in a carbon dioxide incubator for 3 hours, the OD450 value was read with a microplate reader.
数据采用prism7软件进行4参数拟合曲线，最大杀伤计算公式为：1-(最大杀伤孔的OD450值-培养基本底OD450值)/(细胞最大生长孔OD450值-培养基本底OD450值)*100％。结果见表4。The data is fitted with 4 parameters using prism7 software, and the maximum killing formula is: 1-(OD450 value of the maximum killing well-OD450 value of the culture base)/(OD450 value of the cell maximum growth well-OD450 value of the culture base)*100 %. The results are shown in Table 4.
表4喜树碱类化合物活性对比研究结果Table 4 Camptothecin compounds activity comparison research results
Figure PCTCN2022139765-appb-000198
Figure PCTCN2022139765-appb-000198
Figure PCTCN2022139765-appb-000199
Figure PCTCN2022139765-appb-000199
Figure PCTCN2022139765-appb-000200
Figure PCTCN2022139765-appb-000200
以下对具有不同含药连接子的抗体药物偶联物进行活性对比研究。The following is a comparative study on the activity of antibody-drug conjugates with different drug-containing linkers.
实施例5.2不同含药连接子情况下的靶向Her2 ADC的活性对比研究 Example 5.2 Comparative study on the activity of Her2-targeting ADC in the case of different drug-containing linkers
将Her2高表达的细胞系N87(购自：ATCC)从液氮中取出，复苏后用完全培养基将细胞密度调到5×10 4个/ml之后，100μl/孔加入细胞培养板中，过夜培养；使用96孔细胞培养板，对细胞板A排、H排、1列和12列以200μl/孔加入无菌PBS或者无菌纯化水来包边。 Take out the cell line N87 with high expression of Her2 (purchased from: ATCC) from liquid nitrogen, adjust the cell density to 5× 104 cells/ml with complete medium after recovery, add 100 μl/well to the cell culture plate, overnight Cultivation: use 96-well cell culture plates, add 200 μl/well of sterile PBS or sterile purified water to the cell plate row A, H row, column 1 and column 12 to wrap the edges.
使用完全培养基稀释上文制备的靶向Her2的ADC样品，依次稀释到50ug/ml，之后再4倍梯度稀释，共9个梯度加零点，所有样品均设3个复孔。第11列应设阴性对照(细胞+培养基)和空白对照(无细胞，纯培养基)，然后置于细胞培养箱中孵育120h或168小时(详细见表)。取出细胞培养板，40μl/孔加入MTS后，37℃培养箱反应2-4h；取出细胞板，在490nm处读取OD值。The Her2-targeting ADC samples prepared above were diluted with the complete medium, sequentially diluted to 50ug/ml, and then diluted in a 4-fold gradient, with a total of 9 gradients plus a zero point, and three replicate wells were set for all samples. Column 11 should set up a negative control (cells + medium) and a blank control (no cells, pure medium), and then place it in a cell incubator and incubate for 120 hours or 168 hours (see table for details). Take out the cell culture plate, add 40μl/well of MTS, and react in a 37°C incubator for 2-4h; take out the cell plate, and read the OD value at 490nm.
结果见表5。The results are shown in Table 5.
表5.靶向Her2 ADC的体外细胞杀伤效果Table 5. In vitro cell killing effects of Her2-targeted ADCs
Figure PCTCN2022139765-appb-000201
Figure PCTCN2022139765-appb-000201
Figure PCTCN2022139765-appb-000202
Figure PCTCN2022139765-appb-000202
上述结果表明，使用MWD-L1含药连接子的ADC药物的药效优于使用现有MC-GGFG-Dxd、L-A连接子的ADC；而使用自制的桥连含药连接子L-C相较MWD-L1以及MC-GGFG-Dxd要差。The above results show that the drug efficacy of ADC drugs using MWD-L1 drug-containing linker is better than that of ADC using existing MC-GGFG-Dxd and L-A linkers; L1 and MC-GGFG-Dxd are worse.
进一步地，参照上文所述，对使用MWC-L2含药连接子的ADC与使用L-B、MC-GGFG-Dxd的ADC做进一步比较。结果见表6。Further, referring to the above, the ADC using MWC-L2 drug-containing linker was further compared with the ADC using L-B and MC-GGFG-Dxd. The results are shown in Table 6.
表6.靶向Her2 ADC的体外细胞杀伤效果Table 6. In vitro cell killing effects of Her2-targeted ADCs
Figure PCTCN2022139765-appb-000203
Figure PCTCN2022139765-appb-000203
上述结果表明，MWC-L2含药连接子相较已知含药连接子L-B、MC-GGFG-Dxd而言，同样具有一定的体外杀伤优势。The above results show that the MWC-L2 drug-containing linker also has a certain killing advantage in vitro compared with the known drug-containing linkers L-B and MC-GGFG-Dxd.
进一步地，参照上文所述，对MWD-L1含药连接子与MWD-L8、MWD-L9含药连接子进行比较。结果见表7。Further, referring to the above, the drug-containing linker of MWD-L1 was compared with the drug-containing linker of MWD-L8 and MWD-L9. The results are shown in Table 7.
表7.靶向Her2 ADC的体外细胞杀伤效果Table 7. In vitro cell killing effects of Her2-targeted ADCs
Figure PCTCN2022139765-appb-000204
Figure PCTCN2022139765-appb-000204
上述结果表明，含有苯基取代的含药连接子MWD-L1的ADC相较含苯基无取代的含药连接子MWD-L8以及苯基三氟甲基取代的含药连接子MWD-L9的ADC而言，药效更为显著。The above results show that the ADC containing phenyl-substituted drug-containing linker MWD-L1 compared with the ADC containing phenyl-unsubstituted drug-containing linker MWD-L8 and phenyltrifluoromethyl-substituted drug-containing linker MWD-L9 For ADC, the drug effect is more significant.
实施例5.3不同含药连接子情况下的靶向Trop2 ADC的活性对比研究 Example 5.3 Comparative study on the activity of targeting Trop2 ADC in the case of different drug-containing linkers
将Trop2高表达的细胞系BxPC3(购自：ATCC)从液氮中取出，复苏后用完全培养基将细胞密度调到5×10 4个/ml之后，100μl/孔加入细胞培养板中，过夜培养；使用96 孔细胞培养板，对细胞板A排、H排、1列和12列使用200μl/孔加入无菌PBS或者无菌纯化水来包边。 Take out the cell line BxPC3 (purchased from: ATCC) with high Trop2 expression from liquid nitrogen, adjust the cell density to 5× 104 cells/ml with complete medium after recovery, add 100 μl/well to the cell culture plate, overnight Cultivation: use 96-well cell culture plates, add 200 μl/well of sterile PBS or sterile purified water to the cell plate row A, H row, column 1 and column 12 to wrap the edges.
使用完全培养基稀释上文制备的靶向Trop2的ADC样品，依次稀释到50ug/ml，之后再4倍梯度稀释，共9个梯度加零点，所有样品均设3个复孔。第11列应设阴性对照(细胞+培养基)和空白对照(无细胞，纯培养基)，然后置于细胞培养箱中孵育120h。取出细胞培养板，40μl/孔加入MTS后，37℃培养箱反应2-4h；取出细胞板，在490nm处读取OD值。The above-prepared ADC samples targeting Trop2 were diluted with the complete medium, sequentially diluted to 50ug/ml, and then 4-fold serially diluted, a total of 9 gradients plus a zero point, and three replicate wells were set for all samples. Column 11 should set up a negative control (cells + medium) and a blank control (no cells, pure medium), and then place them in a cell incubator and incubate for 120 hours. Take out the cell culture plate, add 40μl/well of MTS, and react in a 37°C incubator for 2-4h; take out the cell plate, and read the OD value at 490nm.
结果见表8。The results are shown in Table 8.
表8.靶向Trop2 ADC的体外细胞杀伤效果Table 8. In vitro cell killing effect of targeting Trop2 ADC
Figure PCTCN2022139765-appb-000205
Figure PCTCN2022139765-appb-000205
Figure PCTCN2022139765-appb-000206
Figure PCTCN2022139765-appb-000206
上述结果表明，使用MWD-L1、MWC-L2含药连接子制备的ADC药物药效相当，且使用本发明含药连接子制备的ADC药物药效均优于使用现有L-B、L-A以及MC-GGFG-Dxd连接子的ADC。The above results show that the drug efficacy of ADC drugs prepared by using MWD-L1 and MWC-L2 drug-containing linkers is equivalent, and the drug efficacy of ADC drugs prepared by using drug-containing linkers of the present invention is better than that of existing L-B, L-A and MC- ADC of the GGFG-Dxd linker.
进一步地，参照上文所述，对Trop2-ADC-1与Trop2-ADC-10以及Trop2-ADC-6做进一步比较。结果见表9。Further, referring to the above, Trop2-ADC-1 was further compared with Trop2-ADC-10 and Trop2-ADC-6. The results are shown in Table 9.
表9.靶向Trop2 ADC的体外细胞杀伤效果Table 9. In vitro cell killing effect of targeting Trop2 ADC
Figure PCTCN2022139765-appb-000207
Figure PCTCN2022139765-appb-000207
通过比较上述实施结果可知，含有不同释药结构的含药连接子的ADC药物存在显著的活性差异，其中使用MWD-L1时的药效最佳。By comparing the above implementation results, it can be seen that there are significant differences in activity of ADC drugs containing drug-containing linkers with different drug release structures, and the drug effect is the best when MWD-L1 is used.
进一步地，参照上文所述，Trop2-ADC-1与Trop2-ADC-9、Trop2-ADC-8做进一步比较。结果见表10。Further, referring to the above, Trop2-ADC-1 was further compared with Trop2-ADC-9 and Trop2-ADC-8. The results are shown in Table 10.
表10.靶向Trop2的ADC体外细胞杀伤效果Table 10. In vitro cell killing effect of ADCs targeting Trop2
Figure PCTCN2022139765-appb-000208
Figure PCTCN2022139765-appb-000208
上述结果表明，本发明提供的新型ADC药物Trop2-ADC-1，相较于Trop2-ADC-8、 Trop2-ADC-9具有一定的活性优势。The above results show that the novel ADC drug Trop2-ADC-1 provided by the present invention has a certain activity advantage compared with Trop2-ADC-8 and Trop2-ADC-9.
实施例5.4不同ADC毒素对于低丰度肿瘤和肿瘤细胞附近低抗原表达细胞的体外药效研究 Example 5.4 In vitro pharmacodynamic study of different ADC toxins on low-abundance tumors and cells with low antigen expression near tumor cells
由于晚期肿瘤的异质性，肿瘤组织的抗原表达不一。低丰度肿瘤，以及肿瘤附近低表达细胞造成的旁观者效应往往影响到晚期肿瘤患者的预后，是药物评估的重要指标。因此对低丰度肿瘤，以及肿瘤细胞附近低表达细胞进行了分别评估。Due to the heterogeneity of advanced tumors, the expression of antigens in tumor tissues varies. Low-abundance tumors and the bystander effect caused by low-expressing cells near the tumor often affect the prognosis of patients with advanced tumors and are important indicators for drug evaluation. Therefore, low-abundance tumors and low-expressing cells near tumor cells were evaluated separately.
(1)不同ADC毒素对于低丰度肿瘤地的体外药效研究(1) In vitro pharmacodynamic study of different ADC toxins on low-abundance tumors
细胞HS-746T购自ATCC；细胞用含10％胎牛血清(FBS)的RPMI 1640/IMEM(1：1)培养液培养，培养液中同时添加青、链霉素，于37℃、含5％CO2空气的培养箱中培养。Cell HS-746T was purchased from ATCC; the cells were cultured with RPMI 1640/IMEM (1:1) culture medium containing 10% fetal bovine serum (FBS), and penicillin and streptomycin were added to the culture medium at the same time, at 37°C, containing 5 cultured in an incubator with % CO2 air.
细胞接种后，细胞经0.1μg/mL和1μg/mL的Trop2-ADC-1、Trop2-ADC-2、Trop2-ADC-5、Trop2-ADC-10、Trop2-ADC-14分别处理168小时后，收集细胞，计数；之后将细胞与Dylight 488 NHS Ester标记的上述Trop2-ADC在4℃避光孵育1小时，离心去上清后以磷酸盐缓冲液(PBS，pH7.4)重悬，用PBS(pH7.4)洗三次，以流式细胞仪BD ACCURI C6 PLUS检测计算Hs746t细胞数。After cell inoculation, the cells were treated with 0.1 μg/mL and 1 μg/mL Trop2-ADC-1, Trop2-ADC-2, Trop2-ADC-5, Trop2-ADC-10, Trop2-ADC-14 respectively for 168 hours, Collect the cells and count them; then incubate the cells with the above-mentioned Trop2-ADC labeled with Dylight 488 NHS Ester at 4°C in the dark for 1 hour, centrifuge to remove the supernatant, resuspend in phosphate buffer (PBS, pH7.4), wash with PBS (pH7.4) was washed three times, and the number of Hs746t cells was calculated by flow cytometer BD ACCURI C6 PLUS.
表11.靶向Trop2的ADC对于低丰度肿瘤HS-746T体外细胞杀伤效果Table 11. The in vitro cell killing effect of ADCs targeting Trop2 on the low-abundance tumor HS-746T
Figure PCTCN2022139765-appb-000209
Figure PCTCN2022139765-appb-000209
通过上述研究可知，对于低丰度肿瘤细胞HS-746T而言。使用含药连接子MWD-L1，MWC-L2及MWF-L6的小分子均表现出一定的杀伤作用。GGFG-DXD、MWD-L7这两种连接形式的ADC对低丰度肿瘤的杀伤作用几乎没有。此外，含有化合物3的含药连接子，相较MWC-1的小分子具有更好的杀伤作用。可能与化合物3本身更良好的细胞膜穿透性有关。From the above studies, it can be seen that for the low-abundance tumor cell HS-746T. Small molecules using drug-containing linkers MWD-L1, MWC-L2 and MWF-L6 all showed certain killing effects. The two linked forms of ADC, GGFG-DXD and MWD-L7, had almost no killing effect on low-abundance tumors. In addition, the drug-containing linker containing compound 3 has a better killing effect than the small molecule of MWC-1. It may be related to the better cell membrane penetration of compound 3 itself.
(2)不同ADC毒素对于肿瘤细胞附近低抗原表达细胞的体外药效研究(2) In vitro pharmacodynamic study of different ADC toxins on cells with low antigen expression near tumor cells
KPL-4细胞、MDA-MB-468细胞购自ATCC，细胞用含10％胎牛血清(FBS)的RPMI 1640/IMEM(1：1)培养液培养，培养液中同时添加青、链霉素，于37℃、含5％CO2空气的培养箱中培养。KPL-4 cells and MDA-MB-468 cells were purchased from ATCC, and the cells were cultured with RPMI 1640/IMEM (1:1) culture medium containing 10% fetal bovine serum (FBS), and penicillin and streptomycin were added to the culture medium at the same time , cultured at 37°C in an incubator containing 5% CO2 air.
HER2阳性的KPL-4细胞和HER2阴性的MDA-MB-468细胞共同接种，或HER2阴性的MDA-MB-468细胞单独接种，经药物处理168小时后，收集细胞，将细胞与Dylight 488 NHS Ester标记的抗HER2抗体在4℃避光孵育1小时，离心去上清后以PBS(pH 7.4)重悬，用PBS洗三次，以流式细胞仪BD ACCURI C6 PLUS检测计算KPL-4和MDA-MB-468细胞比例，并计算各自细胞数。HER2-positive KPL-4 cells and HER2-negative MDA-MB-468 cells were co-inoculated, or HER2-negative MDA-MB-468 cells were inoculated alone, after 168 hours of drug treatment, the cells were collected, and the cells were mixed with Dylight 488 NHS Ester The labeled anti-HER2 antibody was incubated at 4°C in the dark for 1 hour, centrifuged to remove the supernatant, resuspended in PBS (pH 7.4), washed three times with PBS, and calculated by flow cytometer BD ACCURI C6 PLUS to calculate KPL-4 and MDA- MB-468 cell ratio, and calculate the respective cell numbers.
表12.靶向HER2的ADC在HER2阳性的KPL-4细胞和HER2阴性的MDA-MB-468的旁观者效应研究Table 12. Bystander effect study of HER2-targeted ADCs in HER2-positive KPL-4 cells and HER2-negative MDA-MB-468
Figure PCTCN2022139765-appb-000210
Figure PCTCN2022139765-appb-000210
通过上述研究可知，在高浓度组(1μg/mL)的条件下，各ADC组对阴性细胞均具有较好的肿瘤抑制作用。然而，各浓度组阳性细胞数与阴性细胞数的比对可知。MWF-L6及MWC-L2旁观者杀伤作用优于对照组GGFG-DXD以及L-B。It can be seen from the above research that under the condition of high concentration group (1 μg/mL), each ADC group has better tumor inhibitory effect on negative cells. However, the comparison of the number of positive cells and the number of negative cells in each concentration group can be known. The bystander killing effect of MWF-L6 and MWC-L2 was better than that of the control group GGFG-DXD and L-B.
实施例5.5不同含药连接子体内杀伤作用研究 Example 5.5 Study on the killing effect of different drug-containing linkers in vivo
使用人胰腺癌BxPc-3细胞，其购自中国科学院细胞库。BxPc-3细胞用10-cm培养皿贴壁培养，培养条件为RPMI 1640培养液中加10％胎牛血清以及青、链霉素，于37℃，含5％CO2培养箱中培养。一周2-3次传代，当细胞呈指数生长期时，胰酶消化、收集细胞，计数，接种。Human pancreatic cancer BxPc-3 cells, which were purchased from the Cell Bank of the Chinese Academy of Sciences, were used. BxPc-3 cells were cultured in 10-cm culture dishes adherently. The culture conditions were RPMI 1640 medium plus 10% fetal bovine serum, penicillin and streptomycin, and cultured at 37°C in an incubator containing 5% CO2. Subculture 2-3 times a week, when the cells are in the exponential growth phase, trypsinize, collect the cells, count and inoculate.
使用不同含药连接子的抗体药物偶联物Trop2-ADC-1、Trop2-ADC-2、Trop2-ADC-3、Trop2-ADC-5进行不同剂量组条件下分组比较研究(表13)。分别对小鼠静脉注射(IV)给药，给药体积10mL/kg；溶剂组给予相同体积的溶剂(生理盐水)；具体给药剂量和给药方案见表13。每周测2次肿瘤体积，称小鼠体重，记录数据。The antibody-drug conjugates Trop2-ADC-1, Trop2-ADC-2, Trop2-ADC-3, and Trop2-ADC-5 containing different drug linkers were used to conduct group comparison studies under different dosage group conditions (Table 13). The mice were administered intravenously (IV), with an administration volume of 10 mL/kg; the solvent group was administered the same volume of solvent (physiological saline); see Table 13 for the specific dosage and regimen. The tumor volume was measured twice a week, the body weight of the mice was weighed, and the data were recorded.
实验结束、达到实验终点或肿瘤体积达到2000mm 3，CO 2麻醉处死动物，随后解剖取瘤并拍照。结果见表13和图2。 At the end of the experiment, when the end point of the experiment was reached or when the tumor volume reached 2000 mm 3 , the animals were sacrificed under CO 2 anesthesia, and then the tumors were dissected and photographed. The results are shown in Table 13 and Figure 2.
表13.Trop2-ADC-1、Trop2-ADC-2、Trop2-ADC-3、Trop2-ADC-5对人胰腺癌BxPc-3裸小鼠皮下移植瘤的疗效(根据肿瘤体积计算TGI％)。Table 13. The curative effect of Trop2-ADC-1, Trop2-ADC-2, Trop2-ADC-3, Trop2-ADC-5 on human pancreatic cancer BxPc-3 subcutaneously transplanted tumor in nude mice (TGI% calculated according to tumor volume).
Figure PCTCN2022139765-appb-000211
Figure PCTCN2022139765-appb-000211
Figure PCTCN2022139765-appb-000212
Figure PCTCN2022139765-appb-000212
上述结果表明，定点含药连接子MWD-L1、MWC-L2及MWC-L3体内活性均优于对照分子GGFG-DXD。与此同时，尽管MWC-L2与MWC-L3分别采用了VA的二肽结构以及GGFG的四肽释放结构，但体内药效处于一个相当的水平。The above results indicated that the in vivo activities of site-specific drug-containing linkers MWD-L1, MWC-L2 and MWC-L3 were all superior to those of the control molecule GGFG-DXD. At the same time, although MWC-L2 and MWC-L3 adopt the dipeptide structure of VA and the tetrapeptide release structure of GGFG respectively, the drug efficacy in vivo is at a comparable level.
实施例5.6靶向TROP2的抗体药物偶联物体内药效研究 Example 5.6 In vivo pharmacodynamic study of antibody-drug conjugates targeting TROP2
使用人膀胱癌HT1376细胞，其购自中国科学院细胞库。HT1376细胞用10-cm培养皿贴壁培养，培养条件为RPMI 1640培养液中加10％胎牛血清以及青、链霉素，于37℃，含5％CO2培养箱中培养。一周2-3次传代，当细胞呈指数生长期时，胰酶消化、收集细胞，计数，接种。Human bladder cancer HT1376 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. HT1376 cells were cultured in 10-cm culture dishes adherently. The culture conditions were RPMI 1640 culture medium plus 10% fetal bovine serum, penicillin and streptomycin, and cultured at 37°C in an incubator containing 5% CO2. Subculture 2-3 times a week, when the cells are in the exponential growth phase, trypsinize, collect the cells, count and inoculate.
本实施例以抗体药物偶联物Trop2-ADC-14为例，在不同剂量组条件下对Trop2-ADC-14进行分组比较研究(表14)。分别对小鼠静脉注射(IV)给药，给药体积10mL/kg；溶剂组给予相同体积的溶剂(生理盐水)；具体给药剂量和给药方案见表14。每周测2次肿瘤体积，称小鼠体重，记录数据。In this example, taking the antibody-drug conjugate Trop2-ADC-14 as an example, a group comparison study was carried out on Trop2-ADC-14 under different dosage groups (Table 14). The mice were administered intravenously (IV) respectively, with an administration volume of 10 mL/kg; the solvent group was administered the same volume of solvent (physiological saline); see Table 14 for the specific dosage and administration scheme. The tumor volume was measured twice a week, the body weight of the mice was weighed, and the data were recorded.
实验结束、达到实验终点或肿瘤体积达到2000mm 3，CO 2麻醉处死动物，随后解剖取瘤并拍照。结果见表14和图3，示出了各组小鼠肿瘤体积的生长变化情况。 At the end of the experiment, when the end point of the experiment was reached or when the tumor volume reached 2000 mm 3 , the animals were sacrificed under CO 2 anesthesia, and then the tumors were dissected and photographed. The results are shown in Table 14 and Figure 3, which shows the growth changes of tumor volumes in mice in each group.
表14.Trop2-ADC-14对人膀胱癌HT1376裸小鼠皮下移植瘤的疗效(根据肿瘤体积计算TGI％)。Table 14. The curative effect of Trop2-ADC-14 on human bladder cancer HT1376 subcutaneously transplanted tumor in nude mice (TGI% calculated according to tumor volume).
Figure PCTCN2022139765-appb-000213
Figure PCTCN2022139765-appb-000213
注：P值为与溶剂组相比；DS-1062a可商购，也可参照专利公布文件CN105849126A制备Note: The P value is compared with the solvent group; DS-1062a is commercially available, and can also be prepared with reference to the patent publication CN105849126A
上述结果表明，在HT1376人膀胱癌移植瘤模型中，与溶剂组相比，Trop2-ADC-14具有显著肿瘤抑制活性；另一方面，同剂量下，Trop2-ADC-14对肿瘤抑制活性显著优于DS-1062及Trodelvy。The above results showed that in the HT1376 human bladder cancer xenograft model, Trop2-ADC-14 had significant tumor suppressive activity compared with the solvent group; on the other hand, at the same dose, Trop2-ADC-14 had significantly superior tumor suppressive activity. In DS-1062 and Trodelvy.
实施例5.7靶向TROP2的抗体药物偶联物体内药效研究 Example 5.7 In vivo pharmacodynamic study of antibody-drug conjugates targeting TROP2
使用肺癌细胞Calu-3，其购自中国科学院细胞库。Calu-3细胞用10-cm培养皿贴壁培养，培养条件为RPMI 1640培养液中加10％胎牛血清以及青、链霉素，于37℃，含5％CO2培养箱中培养。一周2-3次传代，当细胞呈指数生长期时，胰酶消化、收集细胞，计数，接种。The lung cancer cell Calu-3, which was purchased from the Cell Bank of the Chinese Academy of Sciences, was used. Calu-3 cells were cultured in 10-cm culture dishes adherently. The culture conditions were RPMI 1640 culture medium plus 10% fetal bovine serum, penicillin and streptomycin, and cultured at 37°C in an incubator containing 5% CO2. Subculture 2-3 times a week, when the cells are in the exponential growth phase, trypsinize, collect the cells, count and inoculate.
本实施例以抗体药物偶联物Trop2-ADC-14为例，在不同剂量组条件下对Trop2-ADC-14进行分组比较研究(表15)。分别对小鼠静脉注射(IV)给药，给药体积10mL/kg；溶剂组给予相同体积的溶剂(生理盐水)；具体给药剂量和给药方案见表15。每周测2次肿瘤体积，称小鼠体重，记录数据。In this example, the antibody-drug conjugate Trop2-ADC-14 was taken as an example, and a group comparison study was carried out on Trop2-ADC-14 under different dosage groups (Table 15). The mice were administered intravenously (IV), with an administration volume of 10 mL/kg; the solvent group was administered the same volume of solvent (physiological saline); see Table 15 for the specific dosage and regimen. The tumor volume was measured twice a week, the body weight of the mice was weighed, and the data were recorded.
实验结束、达到实验终点或肿瘤体积达到2000mm 3，CO 2麻醉处死动物，随后解剖取瘤并拍照。结果见表15和图4，示出了各组小鼠肿瘤体积的生长变化情况。 At the end of the experiment, when the end point of the experiment was reached or when the tumor volume reached 2000 mm 3 , the animals were sacrificed under CO 2 anesthesia, and then the tumors were dissected and photographed. The results are shown in Table 15 and Fig. 4, showing the growth changes of the tumor volumes of the mice in each group.
表15.Trop2-ADC-14对人肺癌Calu-3裸小鼠皮下移植瘤的疗效(根据肿瘤体积计算TGI％)。Table 15. The curative effect of Trop2-ADC-14 on human lung cancer Calu-3 subcutaneously transplanted tumor in nude mice (TGI% calculated according to tumor volume).
Figure PCTCN2022139765-appb-000214
Figure PCTCN2022139765-appb-000214
注：P值为与溶剂组相比Note: P value is compared with solvent group
上述结果表明，在Calu-3人肺癌移植瘤模型中，与溶剂组相比，Trop2-ADC-14具有显著肿瘤抑制活性；与此同时，同剂量下，Trop2-ADC-14对肿瘤抑制活性优于DS-1062及Trodelvy TM。 The above results showed that in the Calu-3 human lung cancer xenograft model, Trop2-ADC-14 had significant tumor suppressive activity compared with the solvent group; meanwhile, at the same dose, Trop2-ADC-14 had superior tumor suppressive activity. In DS-1062 and Trodelvy ™ .
实施例5.8靶向TROP2的抗体药物偶联物旁观者效应的研究 Example 5.8 Study on the bystander effect of antibody-drug conjugates targeting TROP2
TROP2阳性的BxPC-3细胞(购自ATCC)和TROP2阴性的HT-29细胞(购自ATCC)共同接种或TROP2阴性的HT-29细胞单独接种于6孔板中。经Trop2-ADC-14(10、30、100ng/mL)或者DS-1062a(100、300ng/mL)处理144小时后，收集细胞，计数。之后将细胞与Dylight 488 NHS Ester标记的Trop2-ADC-14在冰上避光孵育1小时，离心去上清后以PBS重悬，用PBS洗三次，以流式细胞仪BD ACCURI C6 PLUS检测计算BxPC-3和HT-29细胞比例，并计算各自细胞数。结果见图5。TROP2-positive BxPC-3 cells (purchased from ATCC) and TROP2-negative HT-29 cells (purchased from ATCC) were co-seeded or TROP2-negative HT-29 cells were seeded in a 6-well plate alone. After being treated with Trop2-ADC-14 (10, 30, 100 ng/mL) or DS-1062a (100, 300 ng/mL) for 144 hours, the cells were collected and counted. Then incubate the cells with Dylight 488 NHS Ester-labeled Trop2-ADC-14 on ice for 1 hour in the dark, centrifuge to remove the supernatant, resuspend in PBS, wash with PBS three times, and use flow cytometer BD ACCURI C6 PLUS to detect and calculate The ratio of BxPC-3 and HT-29 cells, and calculate the respective cell numbers. The results are shown in Figure 5.
通过上述研究可知，Trop2-ADC-14具有很强的旁观者效应，在杀伤TROP2阳性细胞的同时，杀伤TROP2阴性细胞；Trop2-ADC-14在30ng/mL时旁观者杀伤效应与参比药物DS-1062a在300ng/mL时效果相当(图5中的5A)，结果提示Trop2-ADC-14旁观者效应整体上强于参比药物DS-1062。Trop2-ADC-14、DS-1062a均对单独培养、TROP2阴性的HT-29细胞增殖没有明显影响(图5中的5B)。Through the above studies, it can be seen that Trop2-ADC-14 has a strong bystander effect, killing TROP2-positive cells and killing TROP2-negative cells at the same time; Trop2-ADC-14 has a bystander effect at 30 ng/mL that is comparable to that of the reference drug DS. The effect of -1062a was comparable at 300ng/mL (5A in Figure 5), and the results suggested that the bystander effect of Trop2-ADC-14 was stronger than that of the reference drug DS-1062 as a whole. Both Trop2-ADC-14 and DS-1062a had no significant effect on the proliferation of TROP2-negative HT-29 cells cultured alone (5B in Figure 5).
实施例5.9靶向TROP2的抗体药物偶联物及喜树碱类化合物对不同肿瘤细胞增值抑制的研究 Example 5.9 Antibody Drug Conjugates Targeting TROP2 and Camptothecin Compounds Inhibiting the Growth of Different Tumor Cells
贴壁生长细胞应用SRB法检测抗体药物偶联物或喜树碱类化合物对体外培养肿瘤细胞增殖的影响。接种一定数量的对数生长期细胞于96孔培养板，贴壁生长过夜后，加入不同浓度的抗体、抗体药物偶联物或喜树碱类化合物。144小时后，用三氯乙酸固定。用SRB(以1％冰醋酸配制，浓度为4mg/mL)溶液染色后，每孔加入10mM Tris溶液溶解。用酶标仪在510nm波长下读OD值。The SRB method was used to detect the effects of antibody-drug conjugates or camptothecin compounds on the proliferation of tumor cells cultured in vitro for adherent cells. Inoculate a certain number of cells in the logarithmic growth phase in a 96-well culture plate, and after growing overnight, add different concentrations of antibodies, antibody-drug conjugates or camptothecin compounds. After 144 hours, fix with trichloroacetic acid. After staining with SRB (prepared with 1% glacial acetic acid, concentration 4mg/mL), add 10mM Tris solution to each well to dissolve. Read the OD value with a microplate reader at a wavelength of 510 nm.
抑制率(％)＝(OD值对照孔-OD值给药孔)/OD值对照孔×100％Inhibition rate (%)=(OD value control well-OD value administration well)/OD value control well×100%
悬浮生长细胞应用MTT法检测抗体药物偶联物或喜树碱类化合物对体外培养肿瘤细胞增殖的影响。接种一定数量的对数生长期细胞于96孔培养板，贴壁生长过夜后，加入不同浓度的抗体药物偶联物或喜树碱类化合物。144小时后，每孔加入MTT，继续于37℃5％CO2饱和湿度培养箱中培养4小时。每孔加入100μL三联液，酶标仪上570nm和690nm波长下测定OD值。MTT method was used to detect the effects of antibody-drug conjugates or camptothecin compounds on the proliferation of tumor cells cultured in vitro. A certain number of cells in the logarithmic growth phase were inoculated in a 96-well culture plate, and after the adherent growth overnight, different concentrations of antibody-drug conjugates or camptothecin compounds were added. After 144 hours, MTT was added to each well, and culture was continued for 4 hours at 37° C. in a 5% CO2 saturated humidity incubator. Add 100 μL triple solution to each well, and measure the OD value at 570 nm and 690 nm wavelengths on a microplate reader.
根据各浓度抑制率，以Graphpad Prism 8.0软件计算半数抑制浓度IC50。实验单独重复1次，数据表示为均值±SD。结果见表16。According to the inhibition rate of each concentration, the half inhibitory concentration IC50 was calculated with Graphpad Prism 8.0 software. The experiment was repeated once, and the data were expressed as mean ± SD. The results are shown in Table 16.
表16.靶向TROP2的ADC及喜树碱类化合物对不同肿瘤细胞增值抑制结果Table 16. The results of inhibition of the proliferation of different tumor cells by ADCs targeting TROP2 and camptothecin compounds
Figure PCTCN2022139765-appb-000215
Figure PCTCN2022139765-appb-000215
Figure PCTCN2022139765-appb-000216
Figure PCTCN2022139765-appb-000216
实施例5.10靶向TROP2的抗体药物偶联物内吞功能的研究 Example 5.10 Study on endocytic function of antibody-drug conjugates targeting TROP2
将BxPC-3细胞(购自：ATCC)接种于6孔板中，分别加入荧光标记的1μg/mL Trop2-ADC-14、Trop2-ADC-14mAb(即抗体h23-12)，于37℃分别孵育3、6、12、18、24和48小时。胰酶消化，洗去未内吞药物，然后用流式细胞仪(BD ACCURI C6 PLUS)检测荧光强度。实验单独重复1次。结果见图6。Inoculate BxPC-3 cells (purchased from: ATCC) into 6-well plates, add fluorescently labeled 1 μg/mL Trop2-ADC-14, Trop2-ADC-14mAb (i.e. antibody h23-12) respectively, and incubate at 37° C 3, 6, 12, 18, 24 and 48 hours. Digest with trypsin, wash away non-endocytosed drugs, and then detect the fluorescence intensity with a flow cytometer (BD ACCURI C6 PLUS). The experiment was repeated once alone. The results are shown in Figure 6.
通过上述研究可知，荧光标记的Trop2-ADC-14、h23-12与TROP2阳性细胞BxPC-3温育后，被细胞内吞；內吞具有时间依赖性，随着时间延长內吞药物逐渐增加；Trop2-ADC-14在24小时时的内吞药物大于抗体h23-12。From the above studies, it can be known that fluorescently labeled Trop2-ADC-14 and h23-12 were endocytosed by the cells after incubation with TROP2-positive cells BxPC-3; endocytosis was time-dependent, and the internalized drugs gradually increased with time; Trop2-ADC-14 internalized drug more than antibody h23-12 at 24 hours.
实施例5.11靶向TROP2的抗体药物偶联物及喜树碱类化合物诱导细胞凋亡的研究 Example 5.11 Antibody Drug Conjugates Targeting TROP2 and Camptothecin Compounds Inducing Cell Apoptosis
BxPC-3细胞(购自ATCC)接种于6孔板中，经Trop2-ADC-14(0.020、0.196、1.963nM)、DS-1062(0.020、0.196、1.963nM)、Trop2-ADC-14mAb(即抗体h23-12)(6.728nM)和化合物3(0.1、1、10nM)处理120小时后，收集细胞，加入Annexin-V-FITC和PI，室温避光染色15分钟，最后加入300μl 1x结合缓冲液重悬，用流式细胞仪(BD AccuriTMC6 Plus flow cytometer)检测凋亡，每组样品门中为1×10 4个细胞。实验数据用BD CSamplerTMPlus C6 Plus软件分析。结果见图7。 BxPC-3 cells (purchased from ATCC) were inoculated in 6-well plates, treated with Trop2-ADC-14 (0.020, 0.196, 1.963nM), DS-1062 (0.020, 0.196, 1.963nM), Trop2-ADC-14mAb (ie Antibody h23-12) (6.728nM) and compound 3 (0.1, 1, 10nM) were treated for 120 hours, the cells were collected, Annexin-V-FITC and PI were added, stained at room temperature for 15 minutes in the dark, and finally 300μl 1x binding buffer was added Resuspend and detect apoptosis with a flow cytometer (BD AccuriTMC6 Plus flow cytometer), with 1×10 4 cells in each sample gate. The experimental data were analyzed with BD CSamplerTMPlus C6 Plus software. The results are shown in Figure 7.
通过上述研究可知，Trop2-ADC-14 0.196nM即明显诱导细胞凋亡标志性蛋白pro-PARP降解，诱导pro-caspase 3水解成活性蛋白caspase 3(cleaved-caspase 3)，细胞凋亡诱导作用具有浓度依赖性；Trop2-ADC-14细胞凋亡诱导作用明显强于相同浓度的DS-1062a，后者在0.196nM时凋亡诱导作用并不明显；小分子毒素化合物3同样浓度依赖地诱导BxPC-3细胞凋亡；抗体h23-12对BxPC-3细胞没有明显的凋亡作用。According to the above studies, Trop2-ADC-14 0.196nM can obviously induce the degradation of pro-PARP, a marker protein of apoptosis, and induce the hydrolysis of pro-caspase 3 into active protein caspase 3 (cleaved-caspase 3), and the apoptosis-inducing effect has concentration-dependent; the apoptosis-inducing effect of Trop2-ADC-14 cells was significantly stronger than that of DS-1062a at the same concentration, and the apoptosis-inducing effect of the latter was not obvious at 0.196nM; the small molecule toxin compound 3 also concentration-dependently induced BxPC- 3 cell apoptosis; antibody h23-12 had no obvious apoptosis effect on BxPC-3 cells.
实施例5.12靶向TROP2的抗体药物偶联物与TROP2阳性细胞结合活性的研究 Example 5.12 Study on the binding activity of TROP2-targeting antibody-drug conjugates to TROP2-positive cells
不同浓度(1、3、10、30、100、300、1000、3000、10000、30000、100000ng/mL)荧光标记的Trop2-ADC-14、Trop2-ADC-14mAb(即抗体h23-12)与BxPC-3细胞(购自ATCC)冰上避光孵育1小时，离心去上清后以PBS清洗、重悬，以流式细胞仪(BD ACCURI C6 PLUS)检测与细胞结合的药物荧光强度。实验单独重复1次。结果见表17和图8。Different concentrations (1, 3, 10, 30, 100, 300, 1000, 3000, 10000, 30000, 100000ng/mL) fluorescently labeled Trop2-ADC-14, Trop2-ADC-14mAb (i.e. antibody h23-12) and BxPC -3 cells (purchased from ATCC) were incubated on ice in the dark for 1 hour, centrifuged to remove the supernatant, washed with PBS, resuspended, and the fluorescence intensity of the drug bound to the cells was detected by flow cytometry (BD ACCURI C6 PLUS). The experiment was repeated once alone. The results are shown in Table 17 and Figure 8.
表17.Trop2-ADC-14、h23-12与BxPC-3细胞结合EC50(均数±SD，n＝2)Table 17.Trop2-ADC-14, h23-12 combined with BxPC-3 cells EC50 (mean±SD, n=2)
Figure PCTCN2022139765-appb-000217
Figure PCTCN2022139765-appb-000217
通过上述研究可知，Trop2-ADC-14、抗体h23-12均浓度依赖地与TROP2阳性肿瘤细胞BxPC-3结合，结合EC50分别为1296.0±155.6ng/mL、1137.5±128.0ng/mL，提示二者与TROP2阳性细胞结合能力相当；作为对照，Trop2-ADC-14、抗体h23-12与TROP2阴性细胞HT-29均没有明显结合。结果提示，Trop2-ADC-14、抗体h23-12与肿瘤细胞结合依赖于TROP2的表达水平。Through the above studies, it can be known that Trop2-ADC-14 and antibody h23-12 bind to TROP2-positive tumor cell BxPC-3 in a concentration-dependent manner, and the binding EC50 is 1296.0±155.6ng/mL and 1137.5±128.0ng/mL, respectively, suggesting that both The binding ability is equivalent to that of TROP2-positive cells; as a control, Trop2-ADC-14, antibody h23-12, and TROP2-negative cells HT-29 have no obvious binding. The results suggested that the binding of Trop2-ADC-14 and antibody h23-12 to tumor cells depended on the expression level of TROP2.
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。The above description of the specific embodiments of the present invention does not limit the present invention, and those skilled in the art can make various changes or deformations according to the present invention, as long as they do not depart from the spirit of the present invention, all should belong to the scope of the appended claims of the present invention.

Claims (20)

  1. 结构式I所示的化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药：The compound shown in structural formula I or its pharmaceutically acceptable salt, stereoisomer, solvate or prodrug:
    Figure PCTCN2022139765-appb-100001
    Figure PCTCN2022139765-appb-100001
    结构式I中，R 1、R 2、R 3、R 4独立地为氢、卤素、羟基、C1-6烷氧基、氨基或取代氨基、C1-7烷基或取代的C1-7烷基，或者R 1、R 2、R 3、R 4中的任意两个连同它们所连接的碳原子构成C3-6环状烷基； In structural formula I, R 1 , R 2 , R 3 , and R 4 are independently hydrogen, halogen, hydroxyl, C1-6 alkoxy, amino or substituted amino, C1-7 alkyl or substituted C1-7 alkyl, Or any two of R 1 , R 2 , R 3 , R 4 together with the carbon atoms they are connected to form a C3-6 cyclic alkyl group;
    G为氢、卤素、甲基或甲氧基；G is hydrogen, halogen, methyl or methoxy;
    Y为氧、硫、砜、亚砜、亚甲基或取代亚甲基；Y is oxygen, sulfur, sulfone, sulfoxide, methylene or substituted methylene;
    X为氧或硫；X is oxygen or sulfur;
    n＝0或1。n=0 or 1.
  2. 根据权利要求1所述的化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药，其特征在于，所述化合物为结构式IA所示的化合物：The compound according to claim 1 or its pharmaceutically acceptable salt, stereoisomer, solvate or prodrug, characterized in that, the compound is a compound shown in structural formula IA:
    Figure PCTCN2022139765-appb-100002
    Figure PCTCN2022139765-appb-100002
    结构式IA中，R 1、R 2、R 3、R 4不同时为氢。 In the structural formula IA, R 1 , R 2 , R 3 and R 4 are not hydrogen at the same time.
  3. 结构式II所示的化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药：The compound shown in structural formula II or its pharmaceutically acceptable salt, stereoisomer, solvate or prodrug:
    Figure PCTCN2022139765-appb-100003
    Figure PCTCN2022139765-appb-100003
    结构式II中，R 5为C1-5烷基或由一个或多个取代基取代的C1-5烷基、C3-6环状烷基或由一个或多个取代基取代的C3-6环状烷基、苯基或取代苯基； In the structural formula II, R is C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents Alkyl, phenyl or substituted phenyl;
    G为氢、卤素、甲基或甲氧基；G is hydrogen, halogen, methyl or methoxy;
    X为氧或硫；X is oxygen or sulfur;
    n＝0或1；n=0 or 1;
    当X为氧、G为氢、n＝0时，R 5不可为正丁基。 When X is oxygen, G is hydrogen, and n=0, R 5 cannot be n-butyl.
  4. 根据权利要求3所述的化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药，其特征在于，所述化合物为结构式IIA所示的化合物：The compound according to claim 3 or its pharmaceutically acceptable salt, stereoisomer, solvate or prodrug, characterized in that, the compound is a compound shown in structural formula IIA:
    Figure PCTCN2022139765-appb-100004
    Figure PCTCN2022139765-appb-100004
    结构式IIA中，R 5不可为正丁基。 In structural formula IIA, R 5 cannot be n-butyl.
  5. 根据权利要求1至4中任一项所述的化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药，其特征在于，所述化合物具有如下结构：The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, wherein the compound has the following structure:
    Figure PCTCN2022139765-appb-100005
    Figure PCTCN2022139765-appb-100005
    Figure PCTCN2022139765-appb-100006
    Figure PCTCN2022139765-appb-100006
    Figure PCTCN2022139765-appb-100007
    Figure PCTCN2022139765-appb-100007
    Figure PCTCN2022139765-appb-100008
    Figure PCTCN2022139765-appb-100008
  6. 结构式III所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药：A compound represented by structural formula III or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
    Figure PCTCN2022139765-appb-100009
    Figure PCTCN2022139765-appb-100009
    结构式III中，E选自如下基团，其中，
    Figure PCTCN2022139765-appb-100010
    表示与M的连接位点：     In the structural formula III, E is selected from the following groups, wherein,
    Figure PCTCN2022139765-appb-100010
    Indicates the site of attachment to M:
    Figure PCTCN2022139765-appb-100011
    Figure PCTCN2022139765-appb-100011
    Figure PCTCN2022139765-appb-100012
    Figure PCTCN2022139765-appb-100012
    M为亚苯基或由一个或多个取代基取代的亚苯基，或化学键；在取代的亚苯基中，所述取代基选自烷基、卤代烷基、烷氧基、卤素、酯基、酰胺基和氰基；优选地，M为卤素取代的亚苯基；M is phenylene or phenylene substituted by one or more substituents, or a chemical bond; in substituted phenylene, said substituents are selected from alkyl, haloalkyl, alkoxy, halogen, ester , amido and cyano; preferably, M is a halogen-substituted phenylene;
    SP 1选自C1-8亚烷基、C1-8亚环烷基或C1-21直链亚杂烷基，所述C1-21直链亚杂烷基包含1-11个选自N、O或S的杂原子，其中所述C1-8亚烷基、C1-8亚环烷基和C1-21直链亚杂烷基各自独立地任选被选自羟基、氨基、磺酸基和氰基的一个或多个取代基取代； SP 1 is selected from C1-8 alkylene, C1-8 cycloalkylene or C1-21 straight chain heteroalkylene, the C1-21 straight chain heteroalkylene contains 1-11 selected from N, O Or a heteroatom of S, wherein the C1-8 alkylene, C1-8 cycloalkylene and C1-21 linear heteroalkylene are each independently selected from the group consisting of hydroxyl, amino, sulfonic acid and cyano One or more substituents of the group are substituted;
    SP 2选自-NH(CH2CH2O)aCH2CH2CO-、-NH(CH2CH2O)aCH2CO-、-S(CH2)aCO-或化学键，其中a为1-20的整数，优选1-10的整数，更优选1-6的整数； SP 2 is selected from -NH(CH2CH2O)aCH2CH2CO-, -NH(CH2CH2O)aCH2CO-, -S(CH2)aCO- or a chemical bond, wherein a is an integer of 1-20, preferably an integer of 1-10, more preferably 1- an integer of 6;
    A表示2-4个氨基酸；A means 2-4 amino acids;
    A表示2-4个氨基酸；A means 2-4 amino acids;
    CPT为喜树碱类化合物。CPT is a camptothecin compound.
  7. 根据权利要求6所述的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，其特征在于，所述化合物为结构式IIIA所示的化合物：The compound according to claim 6 or its pharmaceutically acceptable salt, stereoisomer, solvate or prodrug, characterized in that, the compound is a compound shown in structural formula IIIA:
    Figure PCTCN2022139765-appb-100013
    Figure PCTCN2022139765-appb-100013
    结构式IIIA中，R 6、R 7独立地为氢、卤素或Ar’S，Ar’为苯基或由一个或多个取代基取代的苯基，在取代的苯基中，所述取代基选自烷基、烷氧基、卤素、酯基、酰胺基 和氰基； In the structural formula IIIA, R 6 and R 7 are independently hydrogen, halogen or Ar'S, Ar' is phenyl or phenyl substituted by one or more substituents, and in the substituted phenyl, the substituents are selected from alkyl group, alkoxy group, halogen, ester group, amido group and cyano group;
    优选地，Ar’为苯基、4-甲基甲酰基取代苯基或4-甲酰基吗啉取代苯基。Preferably, Ar' is phenyl, 4-methylformyl substituted phenyl or 4-formylmorpholine substituted phenyl.
  8. 根据权利要求6或7所述的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，其特征在于，结构式III或结构式IIIA中，CPT为结构式I所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药：The compound or its pharmaceutically acceptable salt, stereoisomer, solvate or prodrug according to claim 6 or 7, is characterized in that, in structural formula III or structural formula IIIA, CPT is the compound shown in structural formula I or its Pharmaceutically acceptable salts, stereoisomers, solvates or prodrugs:
    Figure PCTCN2022139765-appb-100014
    Figure PCTCN2022139765-appb-100014
    结构式I中，基团G、X、Y、R 1、R 2、R 3、R 4、n与权利要求1中基团G、X、Y、R 1、R 2、R 3、R 4、n的定义相同； In the structural formula I, the groups G, X, Y, R 1 , R 2 , R 3 , R 4 , n and the groups G, X, Y, R 1 , R 2 , R 3 , R 4 , The definition of n is the same;
    优选地，CPT为结构式IA所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药：Preferably, CPT is a compound shown in structural formula IA or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
    Figure PCTCN2022139765-appb-100015
    Figure PCTCN2022139765-appb-100015
    结构式IA中，基团R 1、R 2、R 3、R 4与权利要求2中基团R 1、R 2、R 3、R 4的定义相同，但是R 1、R 2、R 3、R 4可以同时为氢； In the structural formula IA, the groups R 1 , R 2 , R 3 , R 4 have the same definitions as the groups R 1 , R 2 , R 3 , R 4 in claim 2, but R 1 , R 2 , R 3 , R 4 can be hydrogen at the same time;
    在结构式III或结构式IIIA中，结构式I或结构式IA所示的化合物经由其氨基与A的羧基通过酰胺键相连。In structural formula III or structural formula IIIA, the compound represented by structural formula I or structural formula IA is connected to the carboxyl group of A via its amino group through an amide bond.
  9. 根据权利要求6或7所述的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，其特征在于，结构式III或结构式IIIA中，CPT为结构式II所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药：The compound or its pharmaceutically acceptable salt, stereoisomer, solvate or prodrug according to claim 6 or 7, is characterized in that, in structural formula III or structural formula IIIA, CPT is the compound shown in structural formula II or its Pharmaceutically acceptable salts, stereoisomers, solvates or prodrugs:
    Figure PCTCN2022139765-appb-100016
    Figure PCTCN2022139765-appb-100016
    结构式II中，基团G、R 5、X、n与权利要求3中基团G、R 5、X、n的定义相同； In the structural formula II, the groups G, R 5 , X, n are the same as the definitions of the groups G, R 5 , X, n in claim 3;
    优选地，CPT为结构式IIA所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药：Preferably, CPT is a compound shown in structural formula IIA or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
    Figure PCTCN2022139765-appb-100017
    Figure PCTCN2022139765-appb-100017
    结构式IIA中，基团R 5与权利要求4中R 5基团的定义相同，但是可以为正丁基。 In the structural formula IIA, the group R 5 has the same definition as the R 5 group in claim 4, but may be n-butyl.
  10. 根据权利要求6或7所述的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，其特征在于，结构式III或结构式IIIA中，CPT为结构式IV所示的依喜替康(exteacan)衍生物或其药学可接受的盐、立体异构体、溶剂化物或前药：The compound according to claim 6 or 7 or its pharmaceutically acceptable salt, stereoisomer, solvate or prodrug, characterized in that, in structural formula III or structural formula IIIA, CPT is Exiti shown in structural formula IV Exteacan derivatives or pharmaceutically acceptable salts, stereoisomers, solvates or prodrugs thereof:
    Figure PCTCN2022139765-appb-100018
    Figure PCTCN2022139765-appb-100018
    结构式IV中，R 8为氢、三氟甲基、C1-5烷基或由一个或多个取代基取代的C1-5烷基、C3-6环状烷基、或由一个或多个取代基取代的C3-6环状烷基、或卤素； In structural formula IV, R is hydrogen, trifluoromethyl, C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl, or substituted by one or more C3-6 cyclic alkyl group substituted, or halogen;
    在结构式III或结构式IIIA中，结构式IV所示的化合物经由其与R 8连接于同一个碳的羟基与A的羧基通过自释放结构相连。 In structural formula III or structural formula IIIA, the compound represented by structural formula IV is connected to the carboxyl group of A through a self-releasing structure via its hydroxyl group connected to the same carbon as R 8 .
  11. 根据权利要求6至10中任一项所述的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，其特征在于，结构式III或结构式IIIA所示的化合物进一步为结构式V所示的化合物：The compound according to any one of claims 6 to 10 or its pharmaceutically acceptable salt, stereoisomer, solvate or prodrug, characterized in that, the compound shown in structural formula III or structural formula IIIA is further structural formula V Compounds shown:
    Figure PCTCN2022139765-appb-100019
    Figure PCTCN2022139765-appb-100019
    结构式V中，R 6、R 7独立地为Ar’S，Ar’为苯基或由一个或多个取代基取代的苯基； In the structural formula V, R 6 and R 7 are independently Ar'S, and Ar' is phenyl or phenyl substituted by one or more substituents;
    Xh和Yh独立地为氢、卤素、卤代烷基或烷氧基。Xh and Yh are independently hydrogen, halogen, haloalkyl or alkoxy.
  12. 根据权利要求11所述的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，其特征在于，结构式V所示的化合物进一步为结构式V-A所示的化合物：The compound according to claim 11 or its pharmaceutically acceptable salt, stereoisomer, solvate or prodrug, characterized in that, the compound shown in structural formula V is further a compound shown in structural formula V-A:
    Figure PCTCN2022139765-appb-100020
    Figure PCTCN2022139765-appb-100020
    优选地，结构式V-A所示的化合物进一步为结构式V-A-1所示的化合物：Preferably, the compound shown in structural formula V-A is further a compound shown in structural formula V-A-1:
    Figure PCTCN2022139765-appb-100021
    Figure PCTCN2022139765-appb-100021
    或者，结构式V所示的化合物进一步为结构式V-B所示的化合物：Alternatively, the compound shown in structural formula V is further a compound shown in structural formula V-B:
    Figure PCTCN2022139765-appb-100022
    Figure PCTCN2022139765-appb-100022
    优选地，结构式V-B所示的化合物进一步为结构式V-B-1所示的化合物：Preferably, the compound shown in structural formula V-B is further a compound shown in structural formula V-B-1:
    Figure PCTCN2022139765-appb-100023
    Figure PCTCN2022139765-appb-100023
    或者，结构式V所示的化合物进一步为结构式V-C所示的化合物：Alternatively, the compound shown in structural formula V is further a compound shown in structural formula V-C:
    Figure PCTCN2022139765-appb-100024
    Figure PCTCN2022139765-appb-100024
  13. 根据权利要求6至12中任一项所述的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，其特征在于，所述化合物为结构式VI所示的化合物：The compound according to any one of claims 6 to 12 or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, wherein the compound is a compound shown in structural formula VI:
    Figure PCTCN2022139765-appb-100025
    Figure PCTCN2022139765-appb-100025
    优选地，结构式VI所示的化合物进一步为结构式VI-A所示的化合物：Preferably, the compound shown in structural formula VI is further the compound shown in structural formula VI-A:
    Figure PCTCN2022139765-appb-100026
    Figure PCTCN2022139765-appb-100026
    优选地，结构式VI-A所示的化合物进一步为结构式VI-A-1所示的化合物：Preferably, the compound shown in structural formula VI-A is further a compound shown in structural formula VI-A-1:
    Figure PCTCN2022139765-appb-100027
    Figure PCTCN2022139765-appb-100027
    或者，结构式VI所示的化合物进一步为结构式VI-B所示的化合物：Or, the compound shown in structural formula VI is further the compound shown in structural formula VI-B:
    Figure PCTCN2022139765-appb-100028
    Figure PCTCN2022139765-appb-100028
    优选地，结构式VI-B所示化合物进一步为结构式VI-B-1所示的化合物：Preferably, the compound shown in structural formula VI-B is further a compound shown in structural formula VI-B-1:
    Figure PCTCN2022139765-appb-100029
    Figure PCTCN2022139765-appb-100029
    或者，结构式VI所示的化合物进一步为结构式VI-C所示的化合物：Or, the compound shown in structural formula VI is further the compound shown in structural formula VI-C:
    Figure PCTCN2022139765-appb-100030
    Figure PCTCN2022139765-appb-100030
  14. 根据权利要求6至13中任一项所述的化合物或其药学上可接受的盐、立体异构 体、溶剂化物或前药，其特征在于，所述化合物具有如下结构：The compound according to any one of claims 6 to 13 or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, wherein the compound has the following structure:
    Figure PCTCN2022139765-appb-100031
    Figure PCTCN2022139765-appb-100031
    Figure PCTCN2022139765-appb-100032
    Figure PCTCN2022139765-appb-100032
    Figure PCTCN2022139765-appb-100033
    Figure PCTCN2022139765-appb-100033
    Figure PCTCN2022139765-appb-100034
    Figure PCTCN2022139765-appb-100034
    Figure PCTCN2022139765-appb-100035
    Figure PCTCN2022139765-appb-100035
    Figure PCTCN2022139765-appb-100036
    Figure PCTCN2022139765-appb-100036
    Figure PCTCN2022139765-appb-100037
    Figure PCTCN2022139765-appb-100037
    Figure PCTCN2022139765-appb-100038
    Figure PCTCN2022139765-appb-100038
    Figure PCTCN2022139765-appb-100039
    Figure PCTCN2022139765-appb-100039
    Figure PCTCN2022139765-appb-100040
    Figure PCTCN2022139765-appb-100040
  15. 根据权利要求1至14中任一项所述的化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药与抗体或抗体片段制得的抗体药物偶联物；The antibody drug conjugate prepared from the compound according to any one of claims 1 to 14 or its pharmaceutically acceptable salt, stereoisomer, solvate or prodrug and antibody or antibody fragment;
    优选地，所述抗体药物偶联物具有通式
    Figure PCTCN2022139765-appb-100041
    所示结构，其中mAb表示抗体或抗体片段，基团M、SP 1、SP 2、A及CPT与权利要求6至14中任一项中基团M、SP 1、SP 2、A及CPT的定义相同；N为1～10、优选1～8、更优选3～8；     Preferably, the antibody drug conjugate has the general formula
    Figure PCTCN2022139765-appb-100041
    In the structure shown, wherein mAb represents an antibody or an antibody fragment, the groups M, SP 1 , SP 2 , A and CPT are the same as those of the groups M, SP 1 , SP 2 , A and CPT in any one of claims 6 to 14 Same definition; N is 1-10, preferably 1-8, more preferably 3-8;
    其中，E L选自如下基团，其中
    Figure PCTCN2022139765-appb-100042
    表示与mAb中半胱氨酸相连，
    Figure PCTCN2022139765-appb-100043
    表示与M相 连：     Wherein, E L is selected from the following groups, wherein
    Figure PCTCN2022139765-appb-100042
    Indicates that it is linked to cysteine in mAb,
    Figure PCTCN2022139765-appb-100043
    Indicates that it is connected to M:
    E L-1a和/或E L-1b：
    Figure PCTCN2022139765-appb-100044
    和/或
    Figure PCTCN2022139765-appb-100045
    Figure PCTCN2022139765-appb-100046
     EL -1a and/or EL -1b:
    Figure PCTCN2022139765-appb-100044
    and / or
    Figure PCTCN2022139765-appb-100045
    Figure PCTCN2022139765-appb-100046
  16. 根据权利要求15所述的抗体药物偶联物，其特征在于，所述抗体药物偶联物具有通式VII或通式VIII所示的结构：The antibody-drug conjugate according to claim 15, wherein the antibody-drug conjugate has a structure represented by general formula VII or general formula VIII:
    Figure PCTCN2022139765-appb-100047
    Figure PCTCN2022139765-appb-100047
    和/或and / or
    Figure PCTCN2022139765-appb-100048
    Figure PCTCN2022139765-appb-100048
    其中N为1～10、优选1～8、更优选3～8。Wherein N is 1-10, preferably 1-8, more preferably 3-8.
  17. 权利要求15或16的抗体药物偶联物的前药代谢产物，所述前药代谢产物为结构式IX所示的化合物：The prodrug metabolite of the antibody drug conjugate according to claim 15 or 16, said prodrug metabolite is a compound shown in structural formula IX:
    Figure PCTCN2022139765-appb-100049
    Figure PCTCN2022139765-appb-100049
    结构式IX中，基团A、CPT与权利要求6至14中任一项中基团A、CPT的定义相同。In the structural formula IX, the definitions of the groups A and CPT are the same as those of the groups A and CPT in any one of claims 6 to 14.
  18. 根据权利要求17所述的前药代谢产物，其特征在于，所述化合物为结构式IX-A所示的化合物：The prodrug metabolite according to claim 17, wherein the compound is a compound shown in structural formula IX-A:
    Figure PCTCN2022139765-appb-100050
    Figure PCTCN2022139765-appb-100050
    结构式IX-A中，基团A、G、Y、R 1、R 2、R 3、R 4、X、n与权利要求6至14中任一项中基团A、G、Y、R 1、R 2、R 3、R 4、X、n的定义相同； In the structural formula IX-A, the group A, G, Y, R 1 , R 2 , R 3 , R 4 , X, n and the group A, G, Y, R 1 in any one of claims 6 to 14 , R 2 , R 3 , R 4 , X, and n have the same definitions;
    优选地，结构式IX-A所示的化合物进一步为结构式IX-A-1所示的化合物：Preferably, the compound shown in structural formula IX-A is further a compound shown in structural formula IX-A-1:
    Figure PCTCN2022139765-appb-100051
    Figure PCTCN2022139765-appb-100051
    或者，所述化合物为结构式IX-B所示的化合物：Alternatively, the compound is a compound shown in structural formula IX-B:
    Figure PCTCN2022139765-appb-100052
    Figure PCTCN2022139765-appb-100052
    结构式IX-B中，基团A、G、R 5、X、n与权利要求6至14中任一项中基团A、G、R 5、X、n的定义相同。 In the structural formula IX-B, the groups A, G, R 5 , X, n have the same definitions as the groups A, G, R 5 , X, n in any one of claims 6 to 14.
    或者，所述化合物为结构式IX-C所示的化合物：Alternatively, the compound is a compound shown in structural formula IX-C:
    Figure PCTCN2022139765-appb-100053
    Figure PCTCN2022139765-appb-100053
    结构式IX-C中，基团A、R 8与权利要求6至14中任一项中基团A、R 8的定义相同。 In the structural formula IX-C, the groups A and R 8 have the same definitions as the groups A and R 8 in any one of claims 6 to 14.
  19. 权利要求1至18中任一项所述的化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药、抗体药物偶联物或前药代谢产物在制备用于治疗肿瘤的药物中的用途。The compound described in any one of claims 1 to 18 or its pharmaceutically acceptable salt, stereoisomer, solvate or prodrug, antibody drug conjugate or prodrug metabolite is used in the preparation of the drug for treating tumors Uses in medicine.
  20. 采用权利要求1至16中任一项所述的化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药或抗体药物偶联物治疗肿瘤的方法，所述方法包括给有此需要的受试者施用所述化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药或抗体药物偶联物。A method for treating tumors using the compound of any one of claims 1 to 16 or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug or antibody drug conjugate, the method comprising administering A subject in need thereof is administered the compound or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug or antibody drug conjugate thereof.
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